Biomedical subjects

G D Ehrlich

Publications and source records attributed to G D Ehrlich.

At least 37 records · Page 2Linked to original sources

PubMed1998

Analysis of adult otitis media: polymerase chain reaction versus culture for bacteria and viruses.

Recent studies using the polymerase chain reaction (PCR) have identified bacterial and viral genomic sequences in culture-negative pediatric middle ear effusions. To evaluate this technique in adults, 19 effusions were analyzed to compare bacterial and viral culture and PCR detection of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and adenovirus. Effusions from 4 subjects positive for human immunodeficiency virus (HIV) were analyzed by PCR for HIV virus. Three of 19 effusions were culture-positive for bacteria, and 0 of 19 for viruses. Fifteen of 19 effusions were PCR-positive for bacterial genomic sequences, and 0 of 19 for adenovirus. Thirteen of 15 PCR-positive specimens demonstrated S pneumoniae, 5 of 15 H influenzae, and 0 of 13 M catarrhalis. All 4 effusions from HIV-positive subjects were PCR-positive for HIV. No effusion was culture-positive and PCR-negative. These results confirm that culture-negative middle ear effusions contain genomic sequences from bacterial pathogens. Finding of HIV RNA and DNA in effusion from HIV-positives suggests replicating virus in this fluid.

Adenoviridae↗

PubMed1998

Comparative evaluation of culture and PCR for the detection and determination of persistence of bacterial strains and DNAs in the Chinchilla laniger model of otitis media.

This study was designed to determine the persistence of culturable bacteria versus DNA in the presence of a middle ear effusion in a chinchilla model of otitis media. Cohorts of animals were either infected with an ampicillin-resistant Haemophilus influenzae strain or injected with a tripartite inoculum consisting of freeze-thawed Streptococcus pneumoniae; pasteurized Moraxella catarrhalis; and DNA from H influenzae. The H influenzae-infected animals displayed culture positivity and polymerase chain reaction positivity through day 35. In the chinchillas infected with the low-copy number inocula of S pneumoniae, DNA was not detectable after day 1 from the co-inoculated pasteurized M catarrhalis bacteria or the purified H influenzae DNA; however, amplifiable DNA from the live low-copy number bacteria persisted through day 21 even though they were not culture-positive past day 3. These results demonstrate that DNA, and DNA from intact but nonviable bacteria, does not persist in an amplifiable form for more than a day in the presence of an effusion; however, live bacteria, while not culturable, persist in a viable state for weeks.

Ampicillin Resistance↗

PubMed1998

Accuracy of a polymerase chain reaction-based assay for detection of pneumococcal bacteremia in children.

OBJECTIVE: To evaluate the utility of a polymerase chain reaction (PCR)-based assay for identifying pneumococcal DNA in the blood of pediatric patients with suspected bacteremia. METHODS: Children evaluated at the Children's Hospital of Pittsburgh who were having blood drawn for culture had an additional 2 to 3 mL of blood (from the same sampling) obtained and placed in a sodium citrate tube for PCR processing (study group). The control group for this study consisted of children having blood drawn for biochemical analysis who were afebrile, well-appearing, and had no recent illnesses. Specimens were frozen at -70 degrees C and then batch-processed for PCR-based analyses with the JM201/202-204 primer/probe set. Amplified products were detected after liquid hybridization format wherein a 32P end-labeled probe was annealed to the amplified DNA and visualized by autoradiographic analysis after gel retardation. RESULTS: Four hundred eighty study group patients and 103 controls had specimens tested by both PCR and blood culture. Twenty-six (5%) patients had a positive blood culture for a pathogenic organism (21 of which were Streptococcus pneumoniae). Twelve (57%) of the 21 patients with blood cultures positive for S pneumoniae also were positive by PCR. In addition, 206 study group patients and 16 controls with negative blood cultures had positive PCR results. A greater proportion of study group patients were PCR-positive/culture-negative than were controls (206/459 vs 16/103). CONCLUSION: Although this assay currently lacks adequate sensitivity and specificity for clinical use, the high frequency of PCR-positive cases in patients with suspected bacteremia may indicate a greater role for S pneumoniae than had previously been appreciated. Further refinement of this assay as well as the development of a rapid PCR-based assay appears warranted.

Adolescent↗

PubMed1997

Loss of T cell receptor Vbeta repertoires in HIV type 1-infected SCID-hu mice.

Late-stage HIV-1 disease in humans has been associated with perturbations of the T cell receptor (TCR) Vbeta repertoire. It is not known if the observed loss of certain Vbeta families is attributable directly to HIV-1 infection or whether this is a consequence of multiple opportunistic infections. Putative HIV-1-associated superantigens have been postulated to be the cause of the perturbed TCR Vbeta repertoire and the subsequent CD4+ T cell depletion in HIV-1-infected humans. In this study, we examined the human TCR Vbeta repertoire in SCID-hu mice, housed in a pathogen-free environment and infected with a molecularly cloned virus strain, to ascertain directly the effect of HIV-1 on the human TCR Vbeta repertoire in the absence of other infectious agents. We demonstrate that mock-infected human thymus/liver (Thy/Liv) implants in SCID-hu mice have complete TCR Vbeta repertoires, reflective of a normal human thymus. However, HIV-1-infected implants in SCID-hu mice had depleted TCR Vbeta repertoires, corresponding with thymocyte depletion. These results indicate that HIV-1-specific mechanisms are the cause of the TCR Vbeta repertoire depletion in infected implants. However, these thymocyte depletions were not restricted to specific TCR Vbeta subsets. These results are not consistent with the hypothesis that HIV-1 acts as a superantigen in vivo. The disruption of the TCR Vbeta repertoire in the human Thy/Liv implants of the SCID-hu mice suggests that HIV-1 infection may be influencing T cell development in the thymus, contributing to both the overall CD4+ T cell depletion in AIDS and limited TCR repertoire diversity.

Animals↗

PubMed1997

Use of DNA amplification for diagnosis of cytomegalovirus enteritis after intestinal transplantation.

BACKGROUND & AIMS: Intestinal transplantation is a feasible therapy for patients with short-bowel syndrome. However, cytomegalovirus (CMV) enteritis can cause complications. The aim of this study was to investigate the value of polymerase chain reaction (PCR)-based detection methods for CMV in the management of patients with small bowel transplants. METHODS: Comparative evaluation of PCR with histopathology, shell-vial assay, and tube culture of intestinal biopsy specimens was used for the diagnosis of CMV enteritis in 21 patients. RESULTS: Ten patients experienced 21 episodes of CMV enteritis, diagnosed by histopathology, virology, or both. PCR had a sensitivity and specificity of 96% and 69%, respectively, compared with traditional methods, whereas the positive and negative predictive values were 35% and 99%, respectively. Three+ and 4+ signals corresponded to a specificity of 91% and positive predictive value of 59%, respectively. CMV was detected by PCR a median of 11 days (range, 0-32) earlier than other methods and lasted a median of 40 days (range, 21-80) in the 13 episodes, which became PCR-negative and in those patients who developed asymptomatic infection. In 8 episodes, CMV by PCR never became negative and was associated with a relapse of disease confirmed by other methods. CONCLUSIONS: PCR is a sensitive method for the early detection of CMV in intestinal biopsy specimens and can be used for preemptive therapy after intestinal transplantation.

Adult↗

PubMed1997

Mutations in the cationic trypsinogen gene are associated with recurrent acute and chronic pancreatitis.

BACKGROUND & AIMS: We recently identified a single R117H mutation in the cationic trypsinogen gene in several kindreds with an inherited form of acute and chronic pancreatitis (HP1), providing strong evidence that trypsin plays a central role in premature zymogen activation and pancreatitis. However, not all families studied have this mutation. The aim of this study was to determine the disease-causing mutation in kindreds with hereditary pancreatitis that lack the previously identified mutation. METHODS: Clinical features of the HP1 kindreds were compared with those of the new kindreds (HP2), and genetic linkage analysis, screening for mutations through DNA sequencing, and screening an unaffected population were performed. RESULTS: The onset of symptoms was delayed and hospitalizations were fewer in HP2 compared with HP1 (P < 0.05). Linkage of the disease gene to chromosome 7q35 was established (logarithm of the odds, 3.73). Mutational screening identified a single A to T mutation resulting in an asparagine to isoleucine transition mutation at position 21 (N21I) in cationic trypsinogen. The mutation was absent in 94 unrelated individuals, representing 188 unique chromosomes. CONCLUSIONS: The identification of a second mutation in the cationic trypsinogen gene (HP2) suggests a dominant role of trypsin in premature protease activation-mediated forms of acute pancreatitis. The pathogenesis of hereditary pancreatitis also suggests that chronic pancreatitis may result from recurrent acute pancreatitis.

Acute Disease↗

PubMed1997

Distinguishing baboon cytomegalovirus from human cytomegalovirus: importance for xenotransplantation.

The severe shortage of human organs for transplantation is the driving force behind xenotransplant research. Nonhuman primates, particularly baboons, are potential sources of organs and tissues. Human cytomegalovirus (HCMV) is the most common donor-associated infection after allotransplantation. Baboon cytomegalovirus (BCMV) is endemic in baboon populations and therefore is a potential cause of donor-associated disease after xenotransplantation. Accordingly, the ability for BCMV to grow in human cells was determined and a sensitive method to distinguish BCMV from HCMV was developed. Human fibroblasts were permissive for BCMV, isolates exhibited cytopathology characteristic of HCMV, and herpesvirus-like virions were observed by electron microscopy. BCMV and HCMV could be distinguished by restriction fragment length polymorphism patterns and by polymerase chain reaction with primers targeting the BCMV major immediate-early gene promoter. These methods can be used to evaluate BCMV pathogenicity in laboratory and clinical xenotransplant trials.

Animals↗

PubMed1997

Pediatric respiratory papillomatosis: prognostic role of viral typing and cofactors.

Children with recurrent respiratory papillomatosis vary greatly in their clinical disease course. Many have mild disease with eventual remission while others present with an early aggressive airway obstructive course. This study consisted of 24 pediatric patients whose specimens underwent polymerase chain reaction analysis for cytomegalovirus (CMV), herpes simplex virus (HSV), and human papillomavirus (HPV) type. Nineteen of 24 specimens contained enough DNA for this study. None of the specimens were found to contain DNA from HPV-16, -18, -31, -33; CMV; or HSV, which contrasts with our previous findings in adults. Ten patients were infected by HPV-11 and seven of these underwent tracheotomy because of an aggressive tumorigenic clinical course. Nine patients were infected by HPV-6 alone of whom only two required a tracheotomy (P = 0.05, Fisher's Exact Test). The early airway obstructive course associated with HPV-11, however, had no bearing on achieving eventual disease remission, with decannulation achieved in eight of nine children.

Adult↗

PubMed1997

Determination of hepatitis C virus genotypes in the United States by cleavase fragment length polymorphism analysis.

We describe the application of a new DNA-scanning method, which has been termed Cleavase Fragment Length Polymorphism (CFLP; Third Wave Technologies, Inc., Madison, Wis.), for the determination of the genotype of hepatitis C virus (HCV). CFLP analysis results in the generation of structural fingerprints that allow discrimination of different DNA sequences. We analyzed 251-bp cDNA products generated by reverse transcription-PCR of the well-conserved 5'-noncoding region of HCV. We determined the genotypes of 87 samples by DNA sequencing and found isolates representing 98% of the types typically encountered in the United States, i.e., types 1a, 1b, 2a/c, 2b, 3a, and 4. Blinded CFLP analysis of these samples was 100% concordant with DNA sequencing results, such that closely related genotypes yielded patterns with strong familial resemblance whereas more divergent sequences yielded patterns with pronounced dissimilarities. In each case, the aggregate pattern was indicative of genotypic grouping, while finer changes suggested subgenotypic differences. We also assessed the reproducibility of CFLP analysis in HCV genotyping by analyzing three distinct isolates belonging to a single subtype. These three isolates yielded indistinguishable CFLP patterns, as did replicate analysis of a single isolate. This study demonstrates the suitability of this technology for HCV genotyping and suggests that it may provide a low-cost, high-throughput alternative to DNA sequencing or other, more costly or cumbersome genotyping approaches.

Base Sequence↗

PubMed1997

Clinical characteristics of hereditary pancreatitis in a large family, based on high-risk haplotype. The Midwest Multicenter Pancreatic Study Group (MMPSG)

OBJECTIVES: Because there are no markers for hereditary pancreatitis (HP), diagnosis has relied on clinical features and inferences. Identification of the HP disease gene locus on chromosome 7q35 provides the first genetic marker for HP, allowing an accurate comparison of the clinical diagnosis of HP with the presence of a high-risk HP haplotype. Our objectives were to compare the clinical diagnosis of HP with inheritance of the HP gene and to characterize the common clinical features. METHODS: A detailed questionnaire was administered to 102 study participants of a large HP kindred. Blood samples were taken for DNA extraction and high-risk haplotype determination. Clinical findings were compared with the presence of a high-risk haplotype. RESULTS: A family tree of more than 500 members and eight generations was constructed, and clinical features of the 102 participants were determined. HP occurred before the age of 5 yr in 58% of subjects, who presented with common symptoms of abdominal pain, nausea/vomiting, and frequent attacks. Thirty-five probands, of whom 80% had clinical symptoms, carried the high-risk haplotype, confirming previous estimates of 80% penetrance. Thirty-two of the study participants had been clinically diagnosed with HP, whereas 70 were clinically unaffected. With regard to the presence of the high-risk haplotype, 87.5% of the clinically diagnosed patients were affected by HP (true positive), whereas 12.5% did not carry the high-risk haplotype (false positive). Seven obligate carriers were identified through DNA analysis; three had previously been unrecognized because of lack of affected offspring. CONCLUSIONS: The diagnosis of hereditary pancreatitis on clinical grounds alone may be inaccurate in less severe cases, as is the exclusion of carrier status through family tree analysis. Therefore, a definitive diagnosis of hereditary pancreatitis in equivocal cases or exclusion of a carrier state should include analysis of genetic markers.

Chromosomes, Human, Pair 7↗

PubMed1996

Structure and sequence of human FGF8.

Recent evidence indicates that Fgf8 is expressed during vertebrate development in multiple locations involved in the patterning and outgrowth of important embryo structures. Cloning and analysis of the murine gene revealed at least eight potential protein isoforms that share a common carboxyl region, encoded by exons 2 and 3, but possess different amino termini, generated by alternative splicing of RNA encoded by multiple 5' exons (exons 1A, 1B, 1C, and 1D). We now report the cloning and sequence of the human FGF8 gene. Human FGF-8 isoforms are identical to their murine counterparts in the common carboxyl region. Four of the human isoforms are identical to, or very similar to, the murine isoforms in the amino termini. However, four of the potential murine isoforms do not have corresponding human isoforms due to marked sequence divergence, leading to a blocked reading frame in exon 1B of FGF8. The lack of the four murine isoforms in humans raises the question of their function in murine development.

Amino Acid Sequence↗

PubMed1996

Genotypic and phenotypic characterization of a neutralization-resistant breakthrough population of HIV-1.

Certain antibody neutralization escape mutants of HIV-1 map outside of the antibody recognition epitope, thereby suggesting the presence of nonlinear conformational domains. In an effort to begin to define the interacting regions of the HIV envelope proteins, a neutralization-sensitive clone of HIV-1, HXB2/BH10Sal-Bam, was passaged in the presence of the V3-specific monoclonal antibody 0.5beta. DNA sequence analysis of the V3 domain of the breakthrough viral populations revealed one population that retained the parental V3 genotype. Quantitative DNA sequence analysis of this breakthrough population revealed the presence of mutational "hotspots" in several envelope domains that are noncontiguous with V3. Mutations were seen throughout gp41 and the C1, V1/V2, C2, and C5 domains of gp120. In contrast, other regions of gp120, C3, V4, C4, and V5 remained totally unchanged. Within V1, three residues within a 14-amino acid stretch experienced five substitutions and in C5 three residues within a 7-amino acid stretch experienced four substitutions. This finding, that certain residues clustered within particular domains (V1/V2, C5, and gp41) experienced multiple substitutions under a defined environmental stressor, suggests that the degree of adaptive plasticity exhibited by the HIV envelope is limited. Based on this observation it may be possible, using a set of antibodies to various envelope epitopes, to discern a set of rules which explain the interactions of the various envelope domains with each other and with their environment. The insight gained into the physiologic constraints that the envelope proteins are subject to may be useful in developing therapeutic and vaccination strategies.

Antibodies, Monoclonal↗

PubMed1996

Comparative analysis of three nucleic acid-based detection systems for hepatitis C virus RNA in plasma from liver transplant recipients.

The early detection of hepatitis C viraemia (HCV) following liver transplantation is important for monitoring disease recurrence and planning antiviral chemotherapy. In the current study, the sensitivity, specificity, and concordance of three HCV RNA assays were compared using a random sample of 84 plasma specimens from 23 transplant recipients. Two of the assays were prototype commercial tests: Roche Molecular Diagnostic's RT-PCR HCV Amplicor system; and Chiron's Quantiplex HCV-RNA assay. The third was a 'home brew' PCR-liquid hybridization/gel retardation assay developed at the University of Pittsburgh Medical Center (UPMC). On all criteria the PCR-based assays out-performed the Quantiplex assay and displayed an overall concordance of 87%. A high percentage of specimens in the Quantiplex assay gave indeterminate results (12%) or high coefficients of variance (13%). The specificities of all RNA assays were determined using HCV serostatus as the gold standard. Both of the PCR-based assays had specificities of 100%, whereas the Chiron Quantiplex HCV assay had a specificity of 88%, if indeterminates were counted as negatives, and a specificity of 64% if indeterminates were counted as positives. The calculated sensitivities of the PCR-based assays were 56% and 48% for the 'home brew' and the Roche assays, respectively. The UPMC HCV assay, however, was determined to be capable of reproducibly detecting four or fewer chimpanzee infectious doses, suggesting that HCV viraemia was not present in the PCR-negative cases. The sensitivity of the Quantiplex assay was 41% counting indeterminates as negatives and 46% counting them as positives. The high cost of the Quantiplex assay combined with the number of uninterpretable results, the lack of sensitivity, and reduced specificity may limit the usefulness of this assay for monitoring HCV recurrence.

Hepacivirus↗

PubMed1996

Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene.

Hereditary pancreatitis (HP) is a rare, early-onset genetic disorder characterized by epigastric pain and often more serious complications. We now report that an Arg-His substitution at residue 117 of the cationic trypsinogen gene is associated with the HP phenotype. This mutation was observed in all HP affected individuals and obligate carriers from five kindreds, but not in individuals who married into the families nor in 140 unrelated individuals. X-ray crystal structure analysis, molecular modelling, and protein digest data indicate that the Arg 117 residue is a trypsin-sensitive site. Cleavage at this site is probably part of a fail-safe mechanism by which trypsin, which is activated within the pancreas, may be inactivated; loss of this cleavage site would permit autodigestion resulting in pancreatitis.

Arginine↗

PubMed1996

A gene for hereditary pancreatitis maps to chromosome 7q35.

BACKGROUND & AIMS: Hereditary pancreatitis (HP) is an autosomal-dominant disorder with incomplete penetrance characterized by recurrent bouts of severe epigastric pain with onset usually at 5-10 years of age. A genetic linkage study was designed to identify the HP gene. METHODS: A 500-member pedigree was constructed from a U.S. kindred centered in eastern Kentucky and western Virginia. A genome-wide search strategy was employed using a 36-member subset of this family to determine the genetic locus for HP. Testing for linkage to microsatellite loci was performed at 20-cM intervals. RESULTS: Linkage was established between the HP phenotype and chromosome 7q in this subset of the family. Modeled as an autosomal dominant disorder with 80% penetrance, a maximal multipoint logarithm of the odds score of 4.3 was obtained using a four-point analysis consisting of markers D7S684, D7S661, D7S505, and the HP locus. Two microsatellite markers, D7S661 and D7S505, that correspond to the 7q35 region of chromosome 7 spanning a 6-cM region did not evidence obligate recombinations with HP. The centromeric and telomeric limits are defined by recombinations at D7S684 and D7S483, respectively, which generates a 19-cM locus for HP. Utilizing family members from the extended pedigree, a break in the high-risk haplotype between D7S684 and D7S661 was observed, which suggests it may be possible to exclude an additional 8 cM from the HP locus. A maximal pairwise logarithm of the odds score of 4.73 at a recombination fraction of theta at D7S684 was obtained with the addition of these extended family members. CONCLUSIONS: Linkage of HP to 7q35 represents a major advancement in our understanding of the genetic basis of this disorder.

Chromosome Mapping↗

PubMed1996

Time to detection of cytomegalovirus (CMV) DNA in blood leukocytes is a predictor for the development of CMV disease in CMV-seronegative recipients of allografts from CMV-seropositive donors following liver transplantation.

In 35 cytomegalovirus (CMV)-seronegative recipients of livers from CMV-seropositive donors, 32 (91%) developed CMV infection and 24 of them (75%) experienced disease. Polymerase chain reaction for CMV DNA in leukocytes had the best positive and negative predictive values for the development of disease within 2 months from transplantation, and shell-vial or tube culture viremia was the best predictor thereafter. In patients who developed CMV disease, CMV DNA was first detected at 46 days (median; range, 13-128) after transplantation, significantly earlier than the 77 days (range, 46-174) for those who did not develop CMV disease (P = .02). By a semiquantitative method, the CMV DNA level in the first positive sample did not predict disease development. However, the maximum CMV DNA level during infection was significantly higher in patients who developed CMV disease. In CMV-seronegative recipients of livers from CMV-seropositive donors, the time to DNA positivity following transplantation may predict disease progression and be useful as a guide for the initiation of preemptive therapy.

Antibodies, Viral↗

PubMed1996

Multiplex PCR-based assay for detection of Bordetella pertussis in nasopharyngeal swab specimens.

A multiplex PCR-based assay was developed for the detection of Bordetella pertussis in nasopharyngeal swab specimens. The assay simultaneously amplified two separate DNA targets (153 and 203 bp) within a B. pertussis repetitive element and a 438-bp target within the beta-actin gene of human DNA (PCR amplification control). PCR products were detected by a sensitive and specific liquid hybridization gel retardation assay. A total of 496 paired nasopharyngeal swab specimens were tested by both the PCR-based assay and culture. Although 30 (6%) of the specimens inhibited the amplification of the beta-actin target, in all 29 specimens studied, the inhibition disappeared on repeat testing or was easily overcome with a 1:8 dilution or less of specimen digest. Of the 495 specimen pairs yielding a final evaluable result by the PCR-based assay, 19.0% were positive by the PCR-based assay, whereas 13.9% were positive by culture (P < 0.0001). After resolving the PCR-positive, culture-negative results by testing an additional aliquot from these specimens by the multiplex PCR-based assay, the PCR-based assay had a sensitivity and specificity of 98.9 and 99.7%, respectively, compared with values of 73.4 and 100%, respectively, for culture. In comparison with patients with culture-confirmed pertussis, those with PCR-positive, culture-negative results were older and more likely to have had prolonged cough, immunization with pertussis vaccine, or treatment with erythromycin. This multiplex PCR-based assay is substantially more sensitive than culture and identifies specimens that contain inhibitors of PCR.

Actins↗

PubMed1995

Identification of a patient with Streptococcus pneumoniae bacteremia and meningitis by the polymerase chain reaction (PCR).

A polymerase chain reaction (PCR) assay based on the penicillin-binding protein gene PBP2B identified the presence of DNA specific for Streptococcus pneumoniae in the serum and CSF of a patient with culture-proven bacteremia and meningitis. Positive signals were seen to dilutions of 1:125 and 1:390,625 for the blood and CSF specimens, respectively. Potential advantages of PCR over conventional culture include exquisite sensitivity, faster results and the ability to identify the organisms by the presence of species-specific DNA even in patients pretreated with antibiotics.

Aminoacyltransferases↗