Biomedical subjects

D H Persing

Publications and source records attributed to D H Persing.

At least 145 records · Page 8Linked to original sources

PubMed1994

Precore and contiguous regions of hepatitis B virus in liver transplantation for end-stage hepatitis B.

BACKGROUND/AIMS: Recurrent hepatitis B virus (HBV) infection is the leading cause of mortality and morbidity after orthotopic liver transplantation (OLT) for HBV-related liver disease, but the extent of viral genetic variation in this setting remains unknown. METHODS: Eight patients who underwent OLT for HBV-related liver disease were studied; 7 had cirrhosis and 1 had fulminant hepatitis. Four patients received long-term hepatitis B immunoglobulin prophylaxis. A 240-base pair fragment (1742-1981) comprising the precore region of HBV was amplified by polymerase chain reaction from sera drawn before OLT and 6, 12, and 24 months after OLT and analyzed. RESULTS: All sera were positive by polymerase chain reaction. Nucleotide sequence variations were congruent within most patients before and after OLT; however, in one patient, substantial sequence variation was observed, suggesting infection with a new HBV strain. No sequence variation associated with a particular outcome could be identified. Two patients harbored HBV variants with a deletion or insertion upstream of the precore messenger RNA initiation site. CONCLUSIONS: Reinfection after OLT can occasionally be caused by HBV strains different from the one present before OLT. Changes within the sequenced region are not predictive of the outcome of reinfection.

Adult↗

PubMed1994

Detection of a genetic locus encoding resistance to rifampin in mycobacterial cultures and in clinical specimens.

The polymerase chain reaction (PCR) and automated DNA sequencing were used to detect a genetic locus, rpoB, associated with rifampin resistance in Mycobacterium tuberculosis (TB) in clinical isolates and directly in clinical specimens. Primers derived from the sequence of a TB rpoB gene fragment were used to amplify DNA from bacterial and mycobacterial isolates. An rpoB-specific PCR product was obtained for five of five TB, seven of eight other mycobacterial species, Nocardia sp., Corynebacterium sp., Streptomyces sp., Actinomyces sp., and Rhodococcus sp., but not for 15 isolates (eight genera) representing usual bacterial flora. Sequence comparison of the amplified rpoB region revealed the occurrence of TB-specific "signature nucleotides" at three positions. PCR yielded amplification products for seven of 16 clinical specimens. Five of the seven contained TB-specific DNA, as well as sequences that predicted rifampin susceptibility in accord with agar dilution results. None of ten specimens that were culture negative for TB yielded TB-specific PCR products. These results with a limited number of clinical specimens demonstrate the feasibility of direct detection by PCR of rifampin-resistant TB in clinical specimens. Such testing may serve as a rapid surrogate test for multidrug-resistant TB in laboratories with PCR and automated sequencing capability.

Base Sequence↗

PubMed1994

Naturally occurring hepatitis B virus mutants with deletions in the core promoter region.

A novel class of hepatitis B virus mutants in patients with chronic hepatitis B is described. The predicted effect of the mutations is to disrupt the X open reading frame. The location of the genetic alterations within the putative precore promoter also suggests that they may ameliorate precore transcription, which would provide an alternate mechanism for HBeAg(-) escape variation. Definitive conclusions regarding the effects of these mutations must await additional in vitro and in vivo studies.

Adult↗

PubMed1994

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. This apparent discrepancy in 19 culture-negative synovial fluid specimens from 18 patients with Lyme arthritis was investigated. In all 19, DNA sequences characteristic of plasmid-encoded genes OspA and OspB were easily detected. However, despite equivalent or even superior analytic sensitivity for detection of cultured organisms, the reactivity of two genomic DNA targets was often weak or absent altogether in the clinical specimens. This apparent overrepresentation of B. burgdorferi plasmid sequences was found exclusively in clinical specimens and not in cultured organisms. The physiologic imbalance of genomic and plasmid DNA reactivity in B. burgdorferi infection may signal an underlying pathogenetic mechanism.

Antigens, Bacterial↗

PubMed1994

Cultivation and phylogenetic characterization of a newly recognized human pathogenic protozoan.

An intraerythrocytic protozoan (WA1) recently isolated from a patient in Washington State was shown to be morphologically identical to Babesia microti but biologically and genetically distinct. Continuous growth of WA1 was established in stationary erythrocyte cultures. Hybridization of a chemiluminescent Babesia-specific DNA probe to Southern blots of restriction enzyme-digested genomic DNA showed that WA1 could be distinguished from other Babesia species that were antigenically cross-reactive (Babesia gibsoni and babesial parasites from desert bighorn sheep, Ovis canadensis nelsoni) or known to infect humans (B. microti, Babesia divergens, and Babesia equi), or both. A 1436-bp portion of the nuclear small subunit rRNA gene of WA1 was sequenced and analyzed. Genetic distance analysis showed that WA1 is most closely related to the canine pathogen B. gibsoni and lies within a phylogenetic cluster with Theileria species and B. equi. The methodology described will be useful for improved diagnosis and identification of human protozoal pathogens.

Animals↗

PubMed1994

Detection of Borrelia burgdorferi DNA in museum specimens of Peromyscus leucopus.

To determine whether Borrelia burgdorferi was enzootic within the United States at the beginning of the 20th century, ear skin samples taken from museum specimens of the white-footed mouse (Peromyscus leucopus) were examined for evidence of spirochetal DNA. In total, 280 samples from mice collected between 1870 and 1919 were analyzed by a nested polymerase chain reaction protocol. Of these, 2 specimens from the vicinity of Dennis, Massachusetts, during 1894 were reproducibly positive for B. burgdorferi OspA sequences. The remaining 278, representing both currently endemic and nonendemic sites, were negative for spirochetal DNA. These studies suggest that the agent of Lyme disease was present in a suitable reservoir host in the United States before the turn of the century and provide evidence against a hypothesis of recent introduction of this zoonotic agent to North America.

Animals↗

PubMed1994

Fate of Borrelia burgdorferi DNA in tissues of infected mice after antibiotic treatment.

Persistence of Borrelia burgdorferi DNA in tissues following antibiotic treatment was evaluated in C3H mice inoculated intradermally with 10(3) B. burgdorferi N40 or sterile medium. Half of the infected mice and all of the uninfected mice were treated with ceftriaxone 15 days after inoculation for 5 days. Ear and urinary bladder samples were collected on days 20, 30, and 60 after inoculation for culture and for extraction of DNA and amplification of specific spirochetal DNA by polymerase chain reaction (PCR). PCR primers were specific for a 280-bp portion of a highly conserved region of the gene encoding outer surface protein (Osp) A of B. burgdorferi and for a 328-bp part of the OspB gene. There was excellent concordance between culture and PCR for ears (35/36 mice) and bladders (33/36). Both tissues became uniformly negative at the earliest interval tested after antibiotic treatment. Thus, the ability to amplify B. burgdorferi DNA quickly disappeared from tissues that had become culture-negative after antibiotic treatment, suggesting that serial study of PCR-positive tissues and fluids may be useful for evaluating the efficacy of antibiotic therapy in human Lyme disease.

Animals↗

PubMed1994

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. burgdorferi DNA in virtually any tissue of an infected mouse. This analysis, which detects DNA from as few as three spirochetes, revealed the presence of B. burgdorferi DNA in many tissues from severely arthritic C3H/HeJ mice as early as 1 week postinfection. The heart, ear, and ankle were particularly heavily infected, although B. burgdorferi DNA was also detected in spleen, liver, brain, kidney, bladder, uterus, and lymph nodes. In contrast, much lower levels of spirochete DNA were detected in tissues of infected BALB/c mice, which develop less severe arthritis when infected with B. burgdorferi than do C3H/HeJ mice. This difference was evident throughout the 5-week analysis. A competitive PCR method allowed determination of the absolute number of spirochete gene sequences in infected tissues. Ankles and hearts from C3H/HeJ mice were found to harbor 10(7) copies of the B. burgdorferi ospA gene, while these tissues from BALB/c mice contained 5- and 10-fold less B. burgdorferi DNA, respectively. The genetic regulation of severe pathology was analyzed by infecting the offspring of a cross between C3H/HeJ and BALB/c mice. The F1 mice developed severe arthritis and contained high levels of Borrelia DNA in the heart and ankle, similar to the C3H/HeJ parent. These findings indicate that susceptibility to severe arthritis is a dominant trait and suggest that it may correlate with high levels of persisting spirochetes. Models of pathology in Lyme disease should take into consideration the fact that severity of pathology may be directly related to the number of organisms in infected tissues.

Animals↗

PubMed1994

Genetic stability of Borrelia burgdorferi recovered from chronically infected immunocompetent mice.

Persistent infection with Borrelia burgdorferi in the presence of a vigorous host immune response has been demonstrated in humans and in animal models of Lyme disease. Long-term persistence of B. burgdorferi was documented recently in our studies of BALB/c and C3H mice infected with cloned and uncloned strains of B. burgdorferi. From mice inoculated with the cloned strain, 11 isolates were recovered from the skin, bladder, and blood after 1 year of infection. Analysis of the genes encoding the major outer surface proteins (OspA and OspB) by restriction digestion and DNA sequencing showed no evidence of point mutations or other small genetic alterations after 1 year. Genomic macrorestriction analysis of whole B. burgdorferi showed no loss or gross alterations of the plasmids encoding OspA, OspB, or OspC. However, in two isolates, loss of a 38-kb plasmid encoding outer surface protein D was noted. Our studies suggest that loss or alteration of the genes encoding OspA and OspB is not a common occurrence during persistent spirochetal infection and that other possible mechanisms, including invasion of immunologically privileged sites, should be actively explored.

Animals↗

PubMed1994

Development of PCR assays to detect ampicillin resistance genes in cerebrospinal fluid samples containing Haemophilus influenzae.

We developed PCR primers specific for the blaTEM and blaROB ampicillin resistance genes. The specificity of the primers was confirmed by testing a series of Escherichia coli isolates containing a variety of ampicillin resistance genes and a series of ampicillin-resistant and ampicillin-susceptible Haemophilus influenzae isolates. There was a perfect correlation between ampicillin MICs, the presence of beta-lactamase (as determined by the nitrocefin test), and the results with the blaTEM and blaROB primers. Isolates of H. influenzae and Streptococcus pneumoniae obtained from 25 frozen cerebrospinal fluid (CSF) specimens were also tested. Four of 14 H. influenzae isolates were positive with the blaTEM primers; none were positive with the blaROB primers. Ampicillin MICs were determined for the H. influenzae isolates, and penicillin MICs were determined for the S. pneumoniae isolates. Only the four PCR-positive H. influenzae isolates had elevated MICs of ampicillin and were beta-lactamase positive. None of the H. influenzae isolates contained the blaROB gene, and none of the S. pneumoniae isolates produced positive reactions with either primer set. We then used universal primers directed to conserved regions of rRNA and a Haemophilus detection probe to identify which of the 25 frozen samples of CSF contained H. influenzae. Fourteen of the 25 CSF specimens were positive for H. influenzae, which correlated with the number of organisms obtained by culture of the CSF samples. Four of the CSF samples were positive with the blaTEM primer set, and these correlated with the four H. influenzae isolates that were positive when tested directly by PCR. The blaTEM assay required the use of native Taq polymerase because Amplitaq preparations were contaminated with vector DNA that contained the blaTEM-1 gene.

Ampicillin Resistance↗

PubMed1994

Multiplex PCR for identification of methicillin-resistant staphylococci in the clinical laboratory.

A multiplex PCR assay for detection of the staphylococcal mecA gene (the structural gene for penicillin-binding protein 2a) was compared with agar dilution and disk diffusion susceptibility test methods for identifying methicillin resistance. The multiplex PCR assay combined two primer sets (mecA and 16S rRNA) in a single reaction. A total of 500 staphylococcal isolates (228 isolates of Staphylococcus aureus and 272 isolates of coagulase-negative staphylococci) from clinical specimens were studied. For S. aureus, 40 of 40 mecA-positive isolates and 4 of 188 mecA-negative isolates were oxacillin resistant (positive and negative predictive values of 100 and 98%, respectively). In 3 of 4 discordant isolates, resistance was due to hyperproduction of beta-lactamase. For coagulase-negative staphylococci, 148 of 159 mecA-positive isolates and 0 of 113 mecA-negative isolates were oxacillin resistant (positive and negative predictive values of 93 and 100%, respectively). Twenty-six isolates were categorized as indeterminate because of the absence of a detectable 16S rRNA product. Four of these 26 isolates contained mecA when retested. The assay is designed to be incorporated into the work flow of the clinical microbiology laboratory and allows for the identification of intrinsic resistance in a timely and reliable manner.

Base Sequence↗

PubMed1994

Acrodermatitis chronica atrophicans in the United States: clinical and histopathologic features of six cases.

Acrodermatitis chronica atrophicans is a chronic cutaneous disease caused by the Lyme disease spirochete Borrelia burgdorferi. Acrodermatitis chronica atrophicans is endemic in some regions of Europe but is only rarely seen in the United States. This report describes the clinical and histopathologic findings in six cases of acrodermatitis chronica atrophicans seen at the Mayo Clinic between 1912 and 1961. Histologic differences between early and late phases of the disease were observed, and multisystemic symptoms consistent with chronic Lyme disease were documented in a subset of the patients. All five patients from whom biographical data were available were European immigrants. Our data suggest that some of the first patients with Lyme disease in the United States came to the Mayo Clinic earlier in this century.

Acrodermatitis↗

PubMed1993

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. PATIENT: A 41-year-old immunocompetent man with an intact spleen who developed a moderately severe case of babesiosis. MEASUREMENTS: Serum specimens from the patient were assayed by indirect immunofluorescent antibody (IFA) testing for reactivity with seven Babesia species and with WA1, which was propagated in hamsters inoculated with his blood. A Babesia-specific, ribosomal-DNA (rDNA) probe was hybridized to Southern blots of restriction-endonuclease-digested preparations of DNA from WA1, Babesia microti, and Babesia gibsoni. Serum specimens from 83 family members and neighbors were assayed for IFA reactivity with WA1 and B. microti. Small mammals and ticks were examined for Babesia infection. RESULTS: The patient's serum had very strong IFA reactivity with WA1, strong reactivity with B. gibsoni (which infects dogs), but only weak reactivity with B. microti. DNA hybridization patterns with the rDNA probe clearly differentiated WA1 from B. gibsoni and B. microti. Four of the patient's neighbors had IFA titers to WA1 of 256. The tick vector and animal reservoir of WA1 have not yet been identified, despite trapping 83 mammals and collecting 235 ticks. CONCLUSIONS: WA1 is morphologically indistinguishable but antigenically and genotypically distinct from B. microti. Some patients elsewhere who were assumed to have been infected with B. microti may have been infected with WA1. Improved serodiagnostic and molecular techniques are needed for characterizing Babesia species and elucidating the epidemiology of babesiosis, an emergent zoonosis.

Adolescent↗

PubMed1993

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. The precore and proximal core regions of HBV from 37 of 39 patients were sequenced. RESULTS: Four patients (10.8%) harbored nonsense mutants likely to produce an HBeAg negative HBV infection; two such mutants had a G to A substitution at position 1896, one lost the precore initiation codon, and one harbored a stop codon immediately downstream of the precore initiation codon. Recovered sequences from seven additional patients displayed silent or missense mutations in these regions. All delta coinfected patients harbored known wild type strains of HBV. A significantly poorer survival was associated with antibody to HBe positivity and presence of nucleotide substitutions in the precore/core region. CONCLUSIONS: The prevalence of precore mutations in 37 patients from the United States was lower than reported elsewhere; only two patients were found to have the G to A transition mutation in the precore region at position 1896. We conclude that HBeAg negative HBV mutants do not play a predominant etiologic role among North American patients with fulminant hepatitis B.

Adenine↗

PubMed1993

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. Clinical microbiologists will successfully negotiate the transition to tests based on nucleic acid chemistry if they are willing to educate and become educated in the new technology.

DNA↗

PubMed1993

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. However, identification of pathogens by this strategy is hampered by the frequent contamination of reagents used for the amplification reaction, in particular Taq polymerase, with exogenous bacterial DNA. Here, we describe detailed investigations on the use of 8-methoxypsoralen and long-wave UV light to eliminate contaminating DNA in polymerase chain reaction reagents. The clinical utility of the developed procedure was demonstrated in a case of paucibacillary osteomyelitis, for which no specific bacterial agent had been cultured.

Bacteria↗

PubMed1993

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. Inactivation of amplified DNA was highly efficient for all three protocols; the IP and UNG protocols eliminated at least to 3 x 10(9) copies of the product. The primer hydrolysis protocol varied in efficiency depending on the number and position of the 3' ribose residues, but inactivation ranged from 10(4) to 10(9) copies. We conclude that with some modifications, all three systems are effective for eliminating amplified DNA products. Routine implementation of at least one method should help to avoid false-positive results because of carryover contamination.

Base Sequence↗

PubMed1993

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. For amplicons of < or = 100 bp, IP-10-induced electrophoretic shifts were related to the guanidine-cytidine (G + C) content of the PCR product; no apparent shift and no inactivation were observed for a 92-bp herpes simplex virus amplicon (G + C content, 65%), whereas the 100-bp human papillomavirus product (G + C content, 42%) showed a concentration-dependent shift (25 to 100 micrograms/ml) in electrophoretic migration and was partially inactivated. UNG effectively controlled amplicon carryover for target DNA of > or = 240 bp; however, this treatment did not inactivate the two amplicons of < or = 100 bp, regardless of the G + C content of the product. Larger products were inactivated efficiently by both methods, regardless of their G + C contents. We concluded that both IP and UNG effectively inactivated PCR amplicons but not short amplicons of < or = 100 bp. We recommend that with the adoption of PCR technology in clinical laboratories, primers should be designed to produce amplicons of at least 240 to 350 bp (depending on G + C content) and that at least one effective method of controlling carryover contamination should be incorporated into each PCR protocol.

Base Composition↗