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Biomedical subjects

D H Persing

Publications and source records attributed to D H Persing.

At least 55 records · Page 3Linked to original sources
PubMed1999

A colorimetric microtiter plate PCR system detects respiratory syncytial virus in nasal aspirates and discriminates subtypes A and B.

We developed a colorimetric microtiter plate (MTP) PCR system for specific detection of the respiratory syncytial virus (RSV) nucleocapsid gene and differentiation of viral subtypes A and B. Of 47 pediatric nasal aspirate specimens, the sensitivity and specificity were 94.4% (17 of 18) and 100% (15 of 15), respectively, when compared with RSV cell culture isolation in HEp-2 cells. An additional 14 specimens positive for adenoviruses, rhinoviruses, influenza, or parainfluenza viruses did not give positive reactions. PCR testing detected a mean of 0.15 (0.01 to 7.00) plaque-forming units of RSV virions. Inhibition of PCR amplification was detected in 33.3% (6/18) of undiluted specimens and could be avoided by a dilution (1:10) of extracted RNA without decreasing test sensitivity. RSV subtype, as determined by allele-specific probes, was identical to that determined by an immunofluorescence assay. These results indicate that the MTP PCR system provides a sensitive and specific test for clinical laboratory diagnosis and simultaneous subgroup classification of RSV infection.

Cell Culture Techniques↗
PubMed1999

Quantitative detection of Borrelia burgdorferi with a microtiter-based competitive polymerase chain reaction assay.

BACKGROUND: The current understanding of the inflammation associated with Lyme disease directly involves infection caused by Borrelia burgdorferi within specific target tissues, accompanied by a significant host immunologic component driving the inflammatory process. The measurement of spirochetal tissue burden may thus be useful for studying animal models of Lyme disease pathogenesis. Widely available methods based on the culture of spirochetes from tissues do not provide quantitative information. METHODS: We developed and evaluated a quantitative-competitive polymerase chain reaction assay based on amplification of the B. burgdorferi flagellin gene. The assay makes use of a competitive internal standard and a commercially available enzyme-linked immunosorbent assay detection kit. RESULTS AND CONCLUSION: The assay clearly discriminated between infected and uninfected mouse tissues, and an accurate quantitation range of 500 to 20,000 spirochetes per milligram of tissue was obtained. C3H mice were shown to harbor greater amounts of spirochetal genomic DNA than BALB/c mice. Normalization of samples by tissue weight and genomic DNA content both provided acceptable results. These data indicate this assay can be used to provide reliable and meaningful measurements of spirochetal infectious burden, which will be extremely useful for the study of Lyme disease pathogenesis in the murine model.

Animals↗
PubMed1999

Ribosomal DNA sequencing as a tool for identification of bacterial pathogens.

Conventional methods for the identification and characterization of clinical isolates of bacterial pathogens sometimes fall short when such isolates exhibit unusual phenotypic profiles. Recent advances in DNA sequencing technology have greatly enhanced the ability of the microbiologist to determine the identity of a bacterial isolate. Given the relative objectivity of DNA sequence information and growing availability of sequence information databases, a significant movement is now afoot to use molecular methods for the identification of clinical pathogens.

Bacteria↗
PubMed1999

Effective use of polymerase chain reaction for diagnosis of central nervous system infections.

Polymerase chain reaction (PCR)-based testing of cerebrospinal fluid (CSF) specimens has become standard for confirmatory diagnosis of central nervous system (CNS) infections; however, these tests increase health care costs. We reviewed 3-year data from 974 consecutive CSF specimens submitted for detection of seven pathogens by PCR. In 1997, 237 of 367 specimens (64.6%) were submitted for multiple tests, compared with 203 of 522 (38.9%) in 1996 and 18 of 85 (21.2%) in 1995. In each year the arrival of new house officers coincided with a peak in multiple testing. Among 732 specimens submitted for herpesvirus detection, results were positive for 24 (4.6%) of 523 specimens with increased leukocyte counts or protein levels. None of 209 specimens with normal leukocyte and protein levels were positive for herpesviruses. None of 471 CSF specimens submitted for Borrelia burgdorferi detection were PCR-positive. Use of protein and leukocytes to screen CSF specimens before employing PCR for herpesvirus detection would save almost one-third of costs without reducing sensitivity.

Central Nervous System Infections↗
PubMed1999

No evidence of hepatitis G virus in fulminant hepatic failure in children.

BACKGROUND: The cause of fulminant hepatic failure in children remains unknown, but a viral origin has been suspected in most cases. The recently discovered blood-borne virus, hepatitis G, has been suggested as a possible causative agent. METHOD: Six consecutive children who underwent liver transplantation for fulminant hepatic failure were studied. The children were tested for hepatitis G virus antibodies and hepatitis G virus RNA by polymerase chain reaction after excluding other causes of fulminant hepatic failure. RESULTS: No evidence of hepatitis G virus infection was found in these patients. CONCLUSION: Hepatitis G virus is unlikely to be a common cause of fulminant hepatic failure in pediatric patients from the upper midwestern United States.

Adolescent↗
PubMed1999

The clinical significance of simultaneous infection with hepatitis G virus in patients with chronic hepatitis C.

OBJECTIVES: Hepatitis G virus (HGV) is a recently discovered member of the flavivirus family that has been associated with acute and chronic hepatitis. HGV infection has been reported to coexist in 10-20% of patients with chronic hepatitis C. The significance of simultaneous infection with HGV and hepatitis C virus (HCV) remains to be clarified, as do the effects on HGV of therapeutic interventions such as interferon treatment or liver transplantation. THE AIMS OF OUR STUDY WERE: 1) to examine the frequency of HGV infection in the settings of liver transplantation and interferon therapy for hepatitis C; and 2) to compare HGV RNA levels before and after liver transplantation or interferon treatment. METHODS: Pre-treatment sera were available in 65 patients with chronic hepatitis C treated with interferon; pretransplant sera were available in 49 patients transplanted for end stage liver disease associated with chronic hepatitis C. Information collected included age, sex, risk factors for hepatitis, concurrent liver disease, patient and allograft survival, biochemical response to interferon, histological activity index, and degree of fibrosis/cirrhosis. HCV genotyping was performed by sequencing the NS-5 region. HGV quantitation was performed using a research-based branched DNA (bDNA) assay with a set of probes directed at the 5' untranslated region. RESULTS: HGV was detected in 10 of 49 patients (20%) before transplant and in 13 of 65 patients (20%) treated with interferon. There was a female predominance among HGV-positive compared with HGV-negative transplant patients (80% vs 20%; p < 0.01), but such a difference was not observed in the interferon-treated group. Hepatic iron concentration was lower in hepatic explants from patients who were HGV-positive than in those who were HGV-negative (318 +/- 145 microg/g dry weight vs 1497 +/- 2202 microg/g dry weight; p = 0.02). HCV exposure after 1980 was more common in the HGV-positive patients than in those who were HGV-negative for the entire study population (10 of 20 [50%] vs 16 of 66 [24%]; p = 0.03), as well as for the nontransplant subgroup (8 of 12 [67%] vs 12 of 39 [31%]; p = 0.03). HGV RNA levels declined at 1 yr after transplant in seven of eight patients. Among nine patients tested during or after interferon treatment, HGV RNA levels declined from pretreatment levels in all and disappeared in three. CONCLUSIONS: Among patients with chronic hepatitis C treated with either interferon or liver transplantation, the frequency of coinfection with HGV is about 20%. HGV may be a more recent virus in the US than HCV. Coinfection with HGV does not appear to affect the likelihood of response to interferon in patients with hepatitis C. Finally, HGV RNA levels appear to decline after both liver transplantation and interferon therapy, suggesting possible suppression by increased HCV replication in the former case, and a possible drug treatment effect in the latter.

Adult↗
PubMed1999

Clinical significance of TT virus infection in patients with chronic hepatitis C.

OBJECTIVE: The TT virus (TTV) is a novel DNA virus that has recently been identified. The clinical significance of TTV infection in patients with chronic hepatitis C has not been determined. The aim of this study was to determine the prevalence and possible role of TTV in a well characterized population with chronic hepatitis C infection. METHODS: Ninety patients with chronic HCV and known time of HCV acquisition were selected from approximately 250 patients followed at our institution. Characteristics including age, sex, histology, and length of disease were recorded. Direct sequencing of the NS5 region was used for HCV genotyping. TTV DNA detection was based on PCR. RESULTS: TTV infection was present in 24 of 90 (27%) HCV patients. Patients were divided into four groups based on stage of disease; chronic hepatitis (CH, 29 patients), compensated cirrhosis (CC, 17 patients), decompensated cirrhosis (DC, 28 patients), and hepatocellular carcinoma (HCC, 16 patients). TTV was present in 2/29 (7%), 2/17 (12%), 11/28 (39%), and 9/16 (56%) in those with CAH, CC, DC, and HCC respectively. TTV was significantly more prevalent among those with advanced disease (DC and HCC) compared to those with stable disease (CH and CC; p = 0.001). Mean age, sex, and the time from exposure to HCV to development of complications were similar in TTV-positive and -negative patients. TTV infection was more common in patients infected with HCV genotype 1b. Univariate analysis showed that length of HCV infection, HCV genotype 1b, and TTV infection were important in predicting the stage of liver disease in HCV patients. However, after adjusting for length of HCV infection, TTV but not HCV genotype was important in predicting the stage of liver disease. CONCLUSIONS: We conclude that 1) TTV infection is common in patients with chronic HCV; 2) TTV infection is more prevalent among patients with advanced HCV-associated liver disease (DC and HCC) than in those with stable disease (CH and CC); and 3) TTV infection is more common in patients with HCV genotype 1b but is independent from genotype in predicting the stage of HCV-associated liver disease.

Base Sequence↗
PubMed1999

Population dynamics of a naturally occurring heterogeneous mixture of Borrelia burgdorferi clones.

Two unique isolates of Borrelia burgdorferi, differing in plasmid content and outer surface protein C expression, were cultured on sequential captures of a single free-living Peromyscus leucopus mouse and were examined for differences in transmissibility. Both isolates were transmissible from inoculated C.B-17 mice to larval Ixodes scapularis ticks and, subsequently, from infected nymphal ticks to C3H/HeJ mice. Plasmid and protein analyses suggested that the original isolates were a mixed population of B. burgdorferi, and cloning by limiting dilution resulted in the identification of two clonal groups. In addition to being heterogeneous in plasmid and genomic macrorestriction analyses, the clones varied with respect to the electrophoretic mobilities and antigenicity of their OspC proteins, as shown by their reactivity to a panel of monoclonal antibodies. Plasmid analysis of sequential isolates from C3H mice experimentally infected with the primary isolate or various mixtures of its subclones showed an apparently random fluctuation in clonal dominance in the majority of mice. Surprisingly, mice infected with each subclone were permissive to superinfection with the heterologous subclone, despite the presence of anti-B. burgdorferi antibodies at the time of the secondary challenge. These results show conclusively that mice captured at Lyme disease enzootic sites may be infected by mixed populations of genetically and antigenically distinct B. burgdorferi clones and that these infections can be acquired by coinfection or by sequential infection. The lack of cross-immunization between clones existing within a naturally occurring population may play a role in the maintenance of the genetic heterogeneity of B. burgdorferi in nature.

Amino Acid Sequence↗
PubMed1999

Role of genotypic analysis of the thymidine kinase gene of herpes simplex virus for determination of neurovirulence and resistance to acyclovir.

Mutations in the thymidine kinase (TK) gene of herpes simplex virus (HSV) have been associated with resistance to acyclovir (ACY) and possible recognition of neurotropic strains. We sequenced a 335-bp segment of the TK gene to determine the frequency of mutations in HSV strains recovered from dermal, genital, and cerebrospinal fluid (CSF) specimens (n = 200; 102 HSV type 1 [HSV-1] 98 HSV-2 strains). Four polymorphic sites were detected in HSV-1 strains; C513T, A528G, C575T, and C672T. Among the polymorphisms, only C575T resulted in a change of amino acid sequence (residue 192, Ala-->Val). For HSV-2 strains, only one polymorphism (G420T) which resulted in an amino acid substitution (residue 139, Leu-->Phe) was detected. Phenotypic determination of resistance to ACY by a plaque reduction assay of 48 HSV isolates was not correlated with the sequence results of 11 strains in that 7 of these with genotypic polymorphisms were susceptible to the drug in vitro. In addition, of 32 ACY-resistant HSV strains, 28 (87.5%) had no polymorphisms detected in the 335-bp amplicon of the TK gene. There was no statistical difference in the frequency of polymorphisms according to the source of the specimens. We conclude that the detection of nucleic acid polymorphisms in a previously implicated 335-bp segment of the TK gene cannot be interpreted as indicative of either ACY resistance or neurotropism of HSV strains from dermal, genital, and CSF sources.

Acyclovir↗
PubMed1999

Direct mecA detection from blood culture bottles by branched-DNA signal amplification.

A branched-DNA (bDNA) signal amplification method was used to detect the mecA gene directly from blood culture broth growing staphylococci. BACTEC blood culture bottles with positive growth indices and containing staphylococcus-like organisms as shown by Gram stain were tested for the presence of the mecA gene. Comparison of test results was done among 225 patients (one blood culture from each patient). Compared with PCR, the sensitivity and specificity of the bDNA method are 100 and 99%, respectively. The bDNA test is carried out in a 96-well format and requires approximately 6 h to perform. Our preliminary results suggest that direct detection of the mecA gene by bDNA signal amplification is (i) sensitive enough to detect mecA directly from blood culture bottles without the requirement for subculture and (ii) as sensitive and specific as the PCR-based method.

Bacteremia↗
PubMed1999

Detection of enzootic babesiosis in baboons (Papio cynocephalus) and phylogenetic evidence supporting synonymy of the genera Entopolypoides and Babesia.

Blood smear evaluation of two baboons (Papio cynocephalus) experiencing acute hemolytic crises following experimental stem cell transplantation revealed numerous intraerythrocytic organisms typical of the genus Babesia. Both animals had received whole-blood transfusions from two baboon donors, one of which was subsequently found to display rare trophozoites of Entopolypoides macaci. An investigation was then undertaken to determine the prevalence of hematozoa in baboons held in our primate colony and to determine the relationship, if any, between the involved species. Analysis of thick and thin blood films from 65 healthy baboons (23 originating from our breeding facility, 26 originating from an out-of-state breeding facility, and 16 imported from Africa) for hematozoa revealed rare E. macaci parasites in 31%, with respective prevalences of 39, 35, and 12%. Phylogenetic analysis of nuclear small-subunit rRNA gene sequences amplified from peripheral blood of a baboon chronically infected with E. macaci demonstrated this parasite to be most closely related to Babesia microti (97.9% sequence similarity); sera from infected animals did not react in indirect fluorescent-antibody tests with Babesia microti antigen, however, suggesting that they represent different species. These results support an emerging view that the genus Entopolypoides Mayer 1933 is synonymous with that of the genus Babesia Starcovici 1893 and that the morphological variation noted among intracellular forms is a function of alteration in host immune status. The presence of an underrecognized, but highly enzootic, Babesia sp. in baboons may result in substantial, unanticipated impact on research programs. The similarity of this parasite to the known human pathogen B. microti may also pose risks to humans undergoing xenotransplantation, mandating effective screening of donor animals.

Animals↗
PubMed1999

Isolation of a new subspecies, Bartonella vinsonii subsp. arupensis, from a cattle rancher: identity with isolates found in conjunction with Borrelia burgdorferi and Babesia microti among naturally infected mice.

Bacteremia with fever due to a novel subspecies of Bartonella vinsonii was found in a cattle rancher. The subspecies shared major characteristics of the genus Bartonella in terms of most biochemical features and cellular fatty acid profile, but it was distinguishable from other subspecies of B. vinsonii by good growth on heart infusion agar supplemented with X factor and by its pattern of enzymatic hydrolysis of peptide substrates. DNA relatedness studies verified that the isolate belonged to the genus Bartonella and that it was genotypically related to B. vinsonii. The highest level of relatedness was observed with recently characterized strains from naturally infected mice that were coinfected with Borrelia burgdorferi and Babesia microti. We propose the name Bartonella vinsonii subsp. arupensis subsp. nov. as the new subspecies to accommodate these human and murine isolates.

Animals↗
PubMed1999

Determination of hepatitis C virus genotype by direct sequence analysis of products generated with the Amplicor HCV test.

Consistent with other members of the family Flaviviridae, hepatitis C virus (HCV) demonstrates a high degree of sequence variation throughout the coding regions of its genome. However, there is a high degree of sequence conservation found within the 5' untranslated region (UTR) of the genome, making this region a target of choice for most nucleic acid amplification-based detection assays. In this study, the Amplicor HCV test, a commercially available assay which detects the 5'UTR, was used for the detection of HCV RNA in 669 serum samples obtained from a cohort of liver transplantation patients. Amplification products obtained from the HCV-positive cases were subjected to direct sequencing and genotyping based upon seven phylogenetically informative regions within the 5'UTR. Of the 669 specimens, 416 (62.2%) tested positive for the presence of HCV RNA. Of these, 372 (89.4%) specimens were successfully classified into 11 HCV genotypes and subtypes after computer-assisted analysis of the sequence data. Forty-four (10.6%) of the HCV RNA-positive specimens were not classifiable, the majority corresponding to low-titer specimens as determined by the Chiron Quantiplex HCV RNA 2. 0 assay. Additional comparative studies targeting the NS-5 region of the viral genome generally confirmed the accuracy and sensitivity of the 5'UTR-based classifications, with the exception of the misclassification of a small number of type 1a cases as type 1b. We conclude that although the high sequence conservation within the 5'UTR results in the misclassification of a small number of HCV subtypes, the overall gains of efficiency, the shorter turnaround time, the inclusion of contamination control measures, and the low rate of test failure compared to that of methods based on the NS-5 gene together constitute significant advantages over other techniques.

5' Untranslated Regions↗
PubMed1999

Viral genotypes as determinants of autoimmune expression in chronic hepatitis C.

OBJECTIVE: To correlate viral genotypes with the immune manifestations of chronic hepatitis C and evaluate the effect of immune features on disease expression and response to antiviral treatment. DESIGN: We undertook a retrospective analysis of 67 patients with chronic hepatitis C. MATERIAL AND METHODS: Patients were selected for study if they had been screened for autoantibodies and concurrent immune diseases and if viral genotyping had been performed or was possible. Concurrent immune manifestations and responses to interferon therapy were determined. RESULTS: Of the 67 patients, 18 (27%) had one or more immune features. Immune manifestations occurred as commonly in patients with genotype 1 as in those with other genotypes (30% versus 14%; P = 0.3). Concurrent immune features did not distinguish patients, and responses to interferon therapy were similar between patients with and those without immune manifestations. None of the 14 patients with concurrent immune diseases or high-titer autoantibodies (serum titers, 1:320 or more) entered remission during interferon treatment. In contrast, 6 of 53 patients without concurrent immune diseases and no or low-titer autoantibodies had treatment-related remission. These differences, however, were not statistically significant (0% versus 11%; P = 0.3). CONCLUSION: Autoantibodies and concurrent immune diseases are not associated with a particular viral genotype, clinical profile, or treatment outcome. Larger studies are necessary for complete assessment of the influence of prominent immune manifestations on treatment response.

Adult↗