Search PubMed⌕ Search

Biomedical subjects

D H Persing

Publications and source records attributed to D H Persing.

At least 91 records · Page 5Linked to original sources
PubMed1998

Comparative evaluation of colorimetric microtiter plate systems for detection of herpes simplex virus in cerebrospinal fluid.

In the past few years, application of the PCR to the detection of herpes simplex virus (HSV) DNA in the cerebrospinal fluid (CSF) from patients with encephalitis and meningitis has become standard laboratory practice. However, from an operational perspective, the true diagnostic value of PCR in this setting is yet to be realized because most laboratories subject the amplification products to lengthy probe hybridization procedures by Southern blotting. As alternatives to Southern blotting, we evaluated colorimetric microtiter plate (MTP) systems from ViroMed Laboratories, Inc. (PrimeCapture), CPG, Inc. (Quanti-PATH), and Incstar Corp. (GEN-ETI-K), in addition to a system developed at the Mayo Clinic with the PCR ELISA system (Boehringer Mannheim Corp.). We tested PCR products from 86 clinical CSF specimens submitted to our Molecular Microbiology Laboratory. The CSF specimens used had to have sufficient volume for comparative analysis. By conventional Southern blotting methods, 54 were positive and 32 were negative for HSV DNA. Compared with Southern blotting, the sensitivity and specificity were 63.0 and 100.0%, respectively, for the PrimeCapture system, 98. 2 and 96.9%, respectively, for the Quanti-PATH system, 98.2 and 100. 0%, respectively, for the GEN-ETI-K system, and 100.0 and 96.9%, respectively, for the Mayo system. All four MTP systems had turnaround times 12 to 24 h less than that for Southern blotting. There were no significant differences in costs or technologist time between the Mayo system and Southern blotting. Other features of the Mayo system include type-specific genotypic identification of HSV and the potential for determination of drug resistance by DNA sequencing. Overall, we found that colorimetric MTP systems were likely to improve test turnaround times and patient care at no additional cost.

Blotting, Southern↗
PubMed1998

Hepatitis C virus genotypes and hepatitis G virus in hemodialysis patients from Syria: identification of two novel hepatitis C virus subtypes.

High prevalence of hepatitis C (HCV) and hepatitis G (HGV) viruses has been reported among hemodialysis patients with substantial heterogeneity of HCV genotypes throughout the world. We studied HCV prevalence, clinical significance, genotype distribution, and HGV coinfection in hemodialysis patients from Syria. Ninety (75%) of 120 screened patients were HCV antibody positive. Forty-nine (87.5%) of 56 HCV antibody-positive patients had HCV RNA detected by the polymerase chain reaction. The HCV genotyping was possible in 37 of 49 patients (76%). The HCV genotype distribution was genotype 1a, seven (19%); genotype 1b, 10 (27%); genotype 4a, 11 (30%); unmatched sequences, nine (24%). Phylogenetic analysis of unmatched sequences indicated that they represent two distinct and novel subtypes of HCV genotype 4. Hepatitis G virus RNA was detected in 29 (59%) of the HCV RNA-positive patients. No differences were identified between patients infected with HCV alone and those coinfected with HGV. These data demonstrate that HCV infection is common in this population with a genotype distribution predominantly made up of types 1 and 4. Coinfection with HGV had no effect on the outcome of HCV infection.

Adolescent↗
PubMed1997

Diagnosis and monitoring of Whipple disease by polymerase chain reaction.

BACKGROUND: Whipple disease is a chronic, multisystem disorder associated with infection with Tropheryma whippelii, an organism that has not yet been grown on artificial media. In some cases, the diagnosis of Whipple disease is uncertain if it is based on histology alone. Although antibiotic regimens of various durations have been used, the disease recurs in about one third of cases. No test for cure is available. OBJECTIVE: To develop a test that is more sensitive and specific than histologic examination to diagnose Whipple disease and monitor the effects of antibiotic therapy. DESIGN: Retrospective, laboratory-based evaluations of stored tissue specimens. PATIENTS: 30 patients with clinically diagnosed, histologically confirmed Whipple disease and 8 patients in whom Whipple disease was clinically suspected but who did not have definitive histologic evidence. MEASUREMENTS: Pretreatment and post-treatment biopsy specimens of the small bowel and lymph node were tested by polymerase chain reaction for the presence of T. whippeli DNA. RESULTS: Results on PCR were positive in 29 of the 30 specimens from patients with histologically confirmed disease (sensitivity, 96.6%; specificity, 100%) and in 7 of the 8 specimens from patients in whom disease was clinically suspected. Small-bowel biopsy specimens were obtained after treatment from 17 patients (median duration of follow-up, 119 months); specimens from 12 of these patients had positive results on PCR. When these cases were correlated with therapeutic outcome, it was found that 7 of the 12 patients had clinical relapse during subsequent follow-up or had never responded to treatment (positive predictive value, 58% [95% CI, 28% to 85%]). In contrast, none of the 5 patients whose post-treatment biopsy specimens had negative results on PCR had relapse (negative predictive value, 100% [CI, 48% to 100%]; P = 0.044). No correlation was found between post-treatment histology and clinical outcome (P > 0.2). CONCLUSIONS: Polymerase chain reaction is highly sensitive and specific when used to confirm the diagnosis of Whipple disease, to identify inconclusive and suspicious cases, and to monitor response to therapy. A negative result on PCR may predict a low likelihood of clinical relapse; a positive test result that remains positive despite therapy may be associated with a poor clinical outcome. Histopathologic evaluation of post-treatment specimens does not predict clinical cure or relapse.

Actinobacteria↗
PubMed1997

The cold zone: a curious convergence of tick-transmitted diseases.

In recent years, investigators have gained an increasing appreciation of the complexity of the Lyme disease transmission cycle with regard to the number of pathogens involved. Babesia microti, a blood parasite that is related to the organism that causes malaria, frequently accompanies the Lyme disease spirochete in the mouse reservoir. Recently, a newly described Ehrlichia species related to Ehrlichia equi has been found to be transmitted by the deer tick. Human infections with these agents alone and in combination are now being described, and the successful treatment of these infections may depend on proper diagnosis. The convergence of these and other organisms on the Lyme disease transmission cycle provides a unique opportunity to study pathogen-pathogen interactions in a naturally occurring model.

Animals↗
PubMed1997

Genetic heterogeneity of Borrelia burgdorferi in the United States.

To examine in detail Borrelia burgdorferi strain diversity in the United States, 186 isolates from human, tick, and rodent sources were analyzed from multiple distinct geographic regions of the United States and abroad. Strains were characterized by genomic macrorestriction analysis and ospA and 23S rDNA gene sequencing followed by phylogenetic analysis. Results indicate that spirochetal isolates from the United States fall into two major divisions and nine or more subdivisions; human isolates fell into five of these subdivisions. Greater genetic diversity was observed among B. burgdorferi isolates from moderate climatic regions, consistent with increased tick vector and reservoir diversity. All of the Borrelia isolates were reactive by ospA polymerase chain reaction except for Borrelia hermsii controls and several tick isolates from the Northeast, which were shown to lack the 49-kb plasmid encoding outer surface protein A (OspA). The data suggest that US B. burgdorferi isolates demonstrate substantial genetic heterogeneity, with regional differences in spirochete populations.

Animals↗
PubMed1997

Effects of formalin fixation and prolonged block storage on detection of hepatitis C virus RNA in liver tissue.

It has been suggested that prolonged formalin fixation and block storage adversely affect hepatitis C virus (HCV) ribonucleic acid (RNA) detection in tissue by reverse transcriptase-polymerase chain reaction (RT-PCR). We attempted to determine whether short-term perfusion fixation (3-5 days) or prolonged formalin storage adversely affects the detection of HCV RNA in paraffin-embedded tissue in comparison with 24-h fixation. Also, we examined the effects of prolonged storage of paraffin blocks on the sensitivity for HCV detection. We performed RT-PCR in formalin-fixed explanted livers from 20 liver allograft recipients known to be HCV positive (10 with specimens stored for 2-4 years and 10 with specimens stored for > 4 years). We compared the results of perioperative needle liver biopsy specimens fixed overnight with liver sections fixed by perfusion for 3-5 days and bulk liver tissue stored in formalin for years (mean, 6.25 years; range, 2-11 years). HCV RNA was detected in 100%, 85%, and 0% of specimens fixed for 24 h, 3-4 days, and years, respectively. We conclude that HCV can be readily detected in tissue fixed by formalin overnight, sensitivity decreases slightly with intermediate-length fixation, and HCV is rendered undetectable by prolonged fixation. In addition, retention of formalin-fixed tissue in paraffin blocks does not affect the sensitivity of HCV detection.

Formaldehyde↗
PubMed1997

T-helper-cell cytokines in the early evolution of murine Lyme arthritis.

Genetic susceptibility to murine Lyme arthritis has been correlated with the dominance of T-helper (Th1)- or Th2-cell-associated cytokines. To determine when commitment of the Th cell phenotype occurs, we examined the kinetics of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production by lymph node T cells of disease-susceptible C3H/HeN and disease-resistant BALB/c mice from days 2 through 30 of infection, a period encompassing the evolution of disease and early regression. BALB/c mice produced more IFN-gamma on day 2 of infection than did C3H/HeN mice, whereas IL-4 was first detected on day 14. In contrast, only IFN-gamma could be detected in C3H/HeN mice, and the levels steadily increased from day 2 to surpass those seen in BALB/c mice by day 14 of infection. Despite the difference in cytokine profiles, both BALB/c and C3H/HeN mice developed comparable arthritis assessed at 14 days of infection. Arthritis regressed by day 30 in BALB/c mice but persisted in C3H/HeN mice. These studies are the first to demonstrate that the Th2 response to Borrelia burgdorferi infection of BALB/c mice is preceded by a Th1 cytokine response. Moreover, the timing of the appearance of IL-4 suggests that its primary effect is not in preventing disease, as suggested by others, but, rather, in hastening the resolution of inflammation. The implications of these findings for the orchestration of host defense against B. burgdorferi infection are discussed.

Animals↗
PubMed1997

Borrelia burgdorferi enzyme-linked immunosorbent assay for discrimination of OspA vaccination from spirochete infection.

Recombinant Lyme disease vaccines based on purified preparations of outer surface protein A (OspA) have been shown to be effective in preventing transmission of Borrelia burgdorferi in experimental animal models and are now being tested in humans. Since the most widely used screening tests for Lyme disease are based on a whole-cell sonicate of B. burgdorferi, serologic false positivity in vaccinated persons could result from reactivity to OspA within the antigen preparation. In order to avoid serologic false positivity in vaccinated subjects, we developed an immunoassay based on a low-passage-number, naturally occurring variant of B. burgdorferi which lacks the plasmid encoding OspA and OspB. The use of an antigen preparation derived from this organism provided sensitive and specific detection of B. burgdorferi seropositivity in experimental animals and in human Lyme disease cases. The OspA-B-negative enzyme-linked immunosorbent assay (ELISA) also appeared to be capable of discriminating the vaccinated state from vaccine failure and natural infection in experimental animals. Sera from human subjects participating in a vaccine trial gave false-positive results with an ELISA based on an OspA-containing strain, but no such reactivity was observed when the OspA-negative ELISA was used. We conclude that low-passage-number OspA-B-negative isolates in immunoassays may become useful for the immunologic discrimination of the vaccinated state, natural infection, and vaccine failure.

Antigens, Surface↗
PubMed1997

Indeterminate results of the second-generation hepatitis C virus (HCV) recombinant immunoblot assay: significance of high-level c22-3 reactivity and influence of HCV genotypes.

A second-generation recombinant immunoblot assay (RIBA 2.0) is used in the United States to confirm infection with hepatitis C virus (HCV) in samples that are anti-HCV (enzyme immunoassay) positive. In some cases, indeterminate results of RIBA 2.0, which are defined as reactivity to a single antigen species or reactivity limited to two proteins derived from the same coding region of the HCV genome, are encountered. This study was performed to establish the significance of indeterminate RIBA 2.0 results in relation to HCV RNA detection, high positivity for the c22-3 band, and the HCV genotype as determined by direct DNA sequencing. Ninety-six samples with indeterminate RIBA 2.0 results were studied. HCV RNA was detected in 21 of 34 (62%) samples with high reactivity to c22-3 and in 8 of 62 (13%) samples with low reactivity to c22-3. The HCV genotype distribution in samples that were RIBA 2.0 indeterminate and HCV RNA positive was significantly different from that in samples of a control group with positive results for both the RIBA 2.0 and HCV PCR. These results suggest that highly positive c22-3 samples are likely to be associated with HCV viremia and that infection with less common HCV genotypes is more commonly associated with indeterminate RIBA 2.0 results.

Antigens, Viral↗
PubMed1997

Molecular evidence and clinical significance of herpesvirus coinfection in the central nervous system.

A total of 60 cerebrospinal fluid (CSF) specimens from patients manifesting symptoms resembling viral central nervous system (CNS) disease were examined for the presence of herpes simplex virus (HSV), human herpesvirus 6 (HHV-6), Epstein-Barr virus (EBV), cytomegalovirus, varicella-zoster virus, Borrelia burgdorferi, and Tropheryma whippelii DNA by PCR. Of 30 specimens which were selected on the basis of HSV DNA positivity, 2 were concomitantly positive for HHV-6 DNA and 1 was positive for EBV DNA. In the three specimens positive for more than one herpesvirus, amplicons generated with virus-specific primer sets hybridized specifically to the corresponding virus-specific probe. Sequence analysis of the two amplified DNA fragments demonstrated that they were derived from distinct herpesviruses. Of 22 patients with clinically diagnosed encephalitis, 2 of 3 patients coinfected with HSV and HHV-6 died, compared to 1 of 19 (5%) patients infected with only HSV. Of 30 CSF specimens that were negative for HSV DNA, EBV DNA was detected in one sample. These data indicated the presence of DNA specific for two distinct herpesviruses in the same CSF specimen, providing molecular evidence that coinfection with this group of viruses may occur in the CNS.

Borrelia burgdorferi Group↗
PubMed1997

Laboratory diagnosis of central nervous system infections with herpes simplex virus by PCR performed with cerebrospinal fluid specimens.

Until recently, the laboratory diagnosis of central nervous system (CNS) infections with herpes simplex virus (HSV) has been limited by poor sensitivity and/or specificity. We assessed the diagnostic utility of PCR for detection of HSV in over 2,100 specimens referred to the Mayo Clinic from August 1993 to May 1996. DNA extracted from cerebrospinal fluid (CSF) samples with IsoQuick was amplified by PCR with oligonucleotide primers directed to the DNA polymerase gene of HSV, yielding a 290-bp amplicon. HSV DNA was detected in 150 (135 by gel electrophoresis, 15 by Southern blotting only) of 2,106 (7.1%) specimens. PCR-positive CNS disease occurred in patients ranging in age from 13 days to 89 years; 59% of the cases occurred in patients between the ages of 30 and 69, and 21 (14%) of the patients were infants. Genotype analysis was not routinely performed; however, amplification of a 335-bp product within the thymidine kinase gene of HSV revealed 13 positions within a span of 80 nucleotides that accurately identified the two serotypes of the virus according to 14 reference strains. We conclude that PCR detection of HSV DNA in CSF specimens should be considered an emerging "gold standard" for the laboratory diagnosis of CNS infections with this virus.

Adolescent↗
PubMed1997

Characterization of an immunoreactive protein from the agent of human granulocytic ehrlichiosis.

A gene that is homologous to the Ehrlichia chaffeensis groEL operon was recovered and characterized by broad-range PCR amplification of whole blood from patients with human granulocytic ehrlichiosis (HGE) and from infected HL60 cell cultures. Sequence analysis of an 820-bp DNA fragment recovered directly from human blood showed 76.5 and 76.3% identity with cognate sequences from E. chaffeensis and Cowdria ruminantium, respectively. Analysis of a 1.6-kb DNA fragment derived from an HGE agent-infected HL60 cell culture indicated a near-complete open reading frame that contained 75.6 and 75.2% sequence identity with the E. chaffeensis and C. ruminantium groEL sequences, respectively. Phylogenetic analysis of this fragment showed that the HGE agent-derived sequence was related to, but distinct from, the sequences of E. chaffeensis and C. ruminantium. Polyvalent antibody responses to a recombinant fusion protein based on the HGE agent groEL homolog were detected in three of three BALB/c mice that were infected by syringe inoculation with a Wisconsin strain of the HGE agent (WI-1) and nine of nine mice infected by Ixodes scapularis (Ixodes dammini) tick inoculation of an isolate from Nantucket Island, Mass. (NCH-1). No response was detected in mice infected with Borrelia burgdorferi or in control BALB/c mice. Further characterization of the sensitivity and specificity of immune responses to this protein will be facilitated by the use of recombinant fusion proteins or peptides based on the HGE agent-specific groEL homolog.

Amino Acid Sequence↗
PubMed1997

Prevalence of granulocytic Ehrlichia infection among white-tailed deer in Wisconsin.

Human granulocytic ehrlichiosis (HGE) is caused by an agent that is nearly indistinguishable from the veterinary pathogens Ehrlichia equi and Ehrlichia phagocytophila. The deer tick, Ixodes scapularis, is a vector of the HGE agent, and the white-tailed deer is the primary host for adult Ixodes ticks. We assessed the distribution of granulocytic Ehrlichia infection among deer living within (Wisconsin) and outside (western and southern Iowa) the geographic range of L. scapularis. Whole-blood samples were tested for HGE 16S ribosomal DNA (rDNA) by PCR, and E. equi antibody was detected by indirect immunofluorescence assay (IFA). Antibody titers of > or = 1:64 were defined as positive, and all positive samples were retested with a second lot of substrate antigen. E. equi antibody was present in 14 (8%) of 187 Wisconsin deer and 0 of 60 Iowa specimens (rate ratio undefined; P = 0.025). An additional 30 serum samples from Wisconsin deer were excluded because IFA results were discrepant between substrate lots. The reciprocal antibody titers ranged from 64 to 512 (geometric mean, 141) for positive samples. PCR results were positive for 27 (15%) of 181 Wisconsin deer. The prevalence of infection in northwestern Wisconsin deer was not significantly different from that in central Wisconsin deer, as determined by IFA and PCR. In two samples that were sequenced, the 16S rDNA was nearly identical to that of the granulocytic Ehrlichia species but distinct from that of Anaplasma marginale. The DNA sequences of the samples differed from the published sequences for E. equi, E. phagocytophila, and the HGE agent by 1 or 2 nucleotides (> or = 99.1% homology) at phylogenetically informative sites. Granulocytic Ehrlichia organisms in deer are widely distributed within the geographic range of L. scapularis in Wisconsin. Deer may serve as useful sentinels for areas where HGE transmission to humans may occur.

Animals↗
PubMed1997

No evidence for Borrelia burgdorferi infection in lesions of morphea and lichen sclerosus et atrophicus in Spain. A prospective study and literature review.

The possible association of Borrelia burgdorferi with morphea and lichen sclerosus et atrophicus has been the focus of research and discussion in dermatology during the last 10 years. To investigate the etiopathogenic role of B. burgdorferi in morphea and lichen sclerosus et atrophicus lesions in Spain, we studied 14 cases: 8 patients with lichen sclerosus et atrophicus and 6 with morphea. For the whole group, a prospective study was performed, including serologic studies by indirect immunofluorescence, histologic evaluation of skin biopsy specimens, culture studies, and polymerase chain reaction with different primers sensitive for detecting virtually all B. burgdorferi strains tested to date. Although one patient with morphea had positive serologic findings at low titer, we were not able to culture or detect borrelial DNA in any of the specimens. These findings do not confirm an association between B. burgdorferi and morphea and lichen sclerosus et atrophicus.

Adolescent↗
PubMed1997

Molecular diagnostics of infectious diseases.

Over the past several years, the development and application of molecular diagnostic techniques has initiated a revolution in the diagnosis and monitoring of infectious diseases. Microbial phenotypic characteristics, such as protein, bacteriophage, and chromatographic profiles, as well as biotyping and susceptibility testing, are used in most routine laboratories for identification and differentiation. Nucleic acid techniques, such as plasmid profiling, various methods for generating restriction fragment length polymorphisms, and the polymerase chain reaction (PCR), are making increasing inroads into clinical laboratories. PCR-based systems to detect the etiologic agents of disease directly from clinical samples, without the need for culture, have been useful in rapid detection of unculturable or fastidious microorganisms. Additionally, sequence analysis of amplified microbial DNA allows for identification and better characterization of the pathogen. Subspecies variation, identified by various techniques, has been shown to be important in the prognosis of certain diseases. Other important advances include the determination of viral load and the direct detection of genes or gene mutations responsible for drug resistance. Increased use of automation and user-friendly software makes these technologies more widely available. In all, the detection of infectious agents at the nucleic acid level represents a true synthesis of clinical chemistry and clinical microbiology techniques.

Animals↗
PubMed1996

Hepatitis C virus genotypes in the United States: epidemiology, pathogenicity, and response to interferon therapy. Collaborative Study Group.

OBJECTIVE: To study 1) the geographic distribution and clinical significance of hepatitis C virus (HCV) genotypes in the United States and 2) the influence of HCV genotypes on response to interferon therapy. DESIGN: Hepatitis C virus genotype was determined in 179 stored serum samples obtained from patients who were positive for antibody to HCV and for HCV RNA by using polymerase chain reaction. SETTING: Tertiary referral centers in four geographic regions of the United States. PATIENTS: Patients who visited medical centers in the Midwest (50 patients), Northeast (42 patients), Southeast (35 patients), and West (52 patients). MEASUREMENTS: Chaotropic lysis and isopropanol precipitation were used to extract RNA from serum. Polymerase chain reaction was done on the NS5 region and was followed by automated direct sequencing and genotyping of desalted amplification products. RESULTS: 104 patients (58%) had subtype 1a; 38 (21%) had subtype 1b; 4 (2%) had subtype 2a; 23 (13%) had subtype 2b; 8 (5%) had subtype 3a; and 2 (1%) had subtype 4a. Examination of the known risk factors for acquiring HCV showed no association between genotype and mode of acquisition (blood transfusion, injection drug use, employment at a health care facility) or histologic findings at presentation (mild active hepatitis, moderately active hepatitis, or cirrhosis). Sixty-eight percent of patients with genotype 1a, 80% of patients with genotype 1b, and 37% of patients with genotype 2a or 2b had severe hepatitis. Thirteen of 46 (28%) patients with genotype 1a and 4 of 15 (26%) patients with genotype 1b had a complete biochemical response after 6 months of interferon therapy. In contrast, 10 of 14 (71%) patients with genotype 2a or 2b had a complete response to interferon therapy. Five of 39 (13%) patients with genotype 1a, 1 of 14 (7%) patients with genotype 1b, and 2 of 11 (18%) patients with genotype 2a or 2b had a sustained biochemical response. CONCLUSIONS: In the United States, HCV genotypes 1a and 1b are the predominant genotypes in patients with chronic hepatitis C. Genotype is not correlated with mode of virus acquisition or with histologic findings at presentation. Patients with HCV genotype 1a or 1b had more severe liver disease and lower rates of response to interferon therapy than did patients with HCV genotype 2a or 2b. These findings may have implications for predicting outcome and selecting patients for interferon treatment.

Adult↗
PubMed1996

Perpetuation of the agent of human granulocytic ehrlichiosis in a deer tick-rodent cycle.

A human-derived strain of the agent of human granulocytic ehrlichiosis, a recently described emerging rickettsial disease, has been established by serial blood passage in mouse hosts. Larval deer ticks acquired infection by feeding upon such mice and efficiently transmitted the ehrlichiae after molting to nymphs, thereby demonstrating vector competence. The agent was detected by demonstrating Feulgen-positive inclusions in the salivary glands of the experimentally infected ticks and from field-derived adult deer ticks. White-footed mice from a field site infected laboratory-reared ticks with the agent of human granulocytic ehrlichiosis, suggesting that these rodents serve as reservoirs for ehrlichiae as well as for Lyme disease spirochetes and the piroplasm that causes human babesiosis. About 10% of host-seeking deer ticks were infected with ehrlichiae, and of these, 20% also contained spirochetes. Cotransmission of diverse pathogens by the aggressively human-biting deer tick may have a unique impact on public health in certain endemic sites.

Animals↗