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Biomedical subjects

J W Shih

Publications and source records attributed to J W Shih.

At least 73 records · Page 4Linked to original sources
PubMed1991

The use of a recombinant immunoblot assay in the interpretation of anti-hepatitis C virus reactivity among prospectively followed patients, implicated donors, and random donors.

Samples from prospectively followed recipients, their respective donors, and a cohort of random donors were used to evaluate the specificity and efficacy of a recombinant immunoblot assay (RIBA) as an adjunct to anti-hepatitis C virus (HCV) testing by enzyme immunoassay (EIA). RIBA reacted (RIBA+) in 100 percent of patients who developed hepatitis associated with anti-HCV seroconversion documented by EIA and in 100 percent of the EIA-positive (EIA+) donors implicated in these cases. In contrast, RIBA reacted in none of 10 recipients who were EIA+ but did not develop hepatitis, in none of 7 EIA+ patients with hepatitis B or cytomegalovirus infection, in 33 percent of EIA+ donors who were not implicated in hepatitis transmission, and in 37 percent of EIA+ random donors. Hence, the vast majority of EIA+ individuals who have ancillary evidence of HCV infection react on RIBA, whereas the majority of EIA+ individuals in low-risk settings do not react (RIBA-negative, or RIBA-). There was a strong association between RIBA reactivity and the presence of a surrogate marker (elevated alanine aminotransferase [ALT] and/or antibody to hepatitis B core antigen); 43 percent of RIBA+ implicated donors had a surrogate marker as compared to none of 14 EIA+, RIBA- donors. Among EIA+ random donors, 77 percent of those with a surrogate marker were RIBA+, as compared with 29 percent of those without a surrogate marker. In addition, in EIA+ donors, RIBA reactivity correlated with the extent of ALT elevation; 86 percent of those with an ALT greater than 135 IU per L were RIBA+ compared with 18 percent of those with an ALT less than 30 IU per L.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood↗
PubMed1991

Strain analysis of hepatitis B virus on the basis of restriction endonuclease analysis of polymerase chain reaction products.

To assess the value of classifying hepatitis B virus (HBV) strains at the genomic level with products from the polymerase chain reaction (PCR), an HBV PCR DNA typing procedure was developed. The design of this method was based on the selective sensitivity of the PCR product to digestion with different restriction endonucleases and on the size of the fragment resulting from a specific nuclease digestion. On the basis of published nucleotide sequences of different HBV subtypes, a set of primers was selected within the preS-S region. One of the primers was 1679F containing 25 nucleotides (5'-GGGTGGAGCCCTCAGGCTCAGGGCA-3'), and the other was 2254R containing 24 nucleotides (5'-GAAGATGAGGCATAGCAGCAGGAT-3'). All of the reactive sera produced the same sized 575-bp identification band. The product was subjected to digestion by a selected panel of restriction endonucleases. By using prototype HBV of known subtype as a model, these restriction nuclease maps of the PCR product were subtype specific. Most adr subtype clinical samples produced predicted PCR DNA restriction nuclease fragmentation in accordance with the nucleotide sequence of the prototype virus. Occasionally, samples of either the adw or the ayw subtype gave discrepant results in which they appeared to be a different subtype or were resistant to the restriction nuclease digestion. This genetic alteration was consistent in the paired specimens from sexual transmission cases. These results show that the described procedures are applicable to HBV strain assessment.

Base Sequence↗
PubMed1990

Identification of an insertion-sequence-like genetic element in the newly recognized human pathogen Mycoplasma incognitus.

Cloned 2.2-kb DNA (plasmid psb-2.2) of Mycoplasma incognitus, a pathogen in AIDS and non-AIDS patients [Lo et al., Am. J. Trop. Med. Hyg. 41 (1989) 364-376; 601-616], contains a 1405-bp genetic element closely resembling bacterial insertion sequence (IS) elements. This IS-like element has 29-bp terminal inverted repeats with seven mismatches, is immediately flanked by 3-bp direct repeats, and has typical stem-and-loop structures at or near both the termini. Two potential open reading frames (ORF-1 and ORF-2) encode 143 amino acids (aa) and 103 aa, respectively, in this IS-like element. Part (57 aa) of the deduced aa sequence of ORF-2 has a significant homology (43%) with the putative transposase of Escherichia coli IS3. In this study, a series of synthetic oligodeoxyribonucleotides each containing a specific sequence of a selected segment in psb-2.2, have been used as probes which reveal that the IS-like element occurs more than ten times in the genome of M. incognitus. This potentially transposable element has many characteristic features in common with bacterial IS elements.

Amino Acid Sequence↗
PubMed1990

Transfusion-transmitted HTLV-I/II infection in patients undergoing open-heart surgery.

Sera from 5,244 blood donations collected between 1979 and 1987 were screened for antibody to HTLV-I with an enzyme immunoassay (EIA) whose result was confirmed with a two-step procedure including Western blot (WB) and radio immunoprecipitation. Of 20 repeatedly reactive samples, two were confirmed positive for HTLV-I infection. These blood units were transfused to patients undergoing cardiac surgery. Both recipients of the confirmed anti-HTLV-I positive units were infected with HTLV-I as evidenced by antibody seroconversion. In contrast, six recipients of EIA positive, WB indeterminate blood and nine recipients WB negative blood were not infected with HTLV-I. These results confirm a low prevalence of HTLV-I infection in US blood donors, the capacity of infected units to transmit the virus to recipients, and the importance of an appropriate confirmatory assay.

Blood Donors↗
PubMed1990

Expression of pre-S2 region of hepatitis B surface antigen in Escherichia coli.

We constructed a recombinant plasmid that can express the entire pre-S2 sequence of hepatitis B surface antigen (HBsAg) as a fusion protein in E. coli. The hybrid protein, which comprises the bacterial TrpLE protein and the pre-S2 sequence, was the prominent protein that was found in cell extracts. As determined by immune blot analysis, this protein reacted with human HBV convalescent sera, as well as with sera from animals immunized with either purified HBsAg or isolated polypeptides containing pre-S2. It bound specifically to 125I-polymerized human albumin cross-linked with glutaraldehyde but not to 125I-monomeric human albumin. A novel adjuvant formulation was used in place of Freund's adjuvant to immunize guinea pigs with the recombinant product. The antisera obtained from serial bleedings were found to react with HBsAg of both d and y subtypes. These antisera were also shown to react solely with HBsAg polypeptides which contain of HBsAg to solid-phase polymerized the binding of HBsAg to solid-phase polymerized human albumin.

Animals↗
PubMed1990

Hepatitis B virus propagated in a rat hepatoma cell line is infectious in a primate model.

The human hepatitis B virus (HBV) produced by a rat hepatoma cell line through transfection with HBV DNA is infectious in the human primate model--chimpanzee. Since hepadnaviruses are known to have an extremely narrow host range, our results support the idea that the host species barrier of HBV infection resides on the penetration/adsorption step rather than any postpenetration intracellular event during the virus life cycle.

Animals↗
PubMed1990

Clinical correlation and genetic polymorphism of the human immunodeficiency virus proviral DNA obtained after polymerase chain reaction amplification.

Polymerase chain reaction (PCR) was used to amplify a long sequence of human immunodeficiency virus (HIV) DNA, to assess the correlation between PCR signal and clinical stage of disease, and to demonstrate the genotypic variability of different HIV isolates. Twenty-four (96%) of 25 anti-HIV-reactive patients and none of 12 controls were positive for HIV proviral DNA by PCR. After quantification of the PCR signal, a significant difference in the relative amount of HIV proviral DNA per 10(5) peripheral blood mononuclear cells between symptomatic patients (Centers for Disease Control [CDC] class IV) (32,284 +/- 5225 cpm [mean +/- SE], equivalent to 802 HIV plasmid DNA copies) and patients without symptoms (CDC class II/III) (5484 +/- 1469 cpm [mean +/- SE], equivalent to 67 HIV plasmid DNA copies) was observed (P less than .01). Restriction analysis of PCR products in selected samples showed extensive genetic polymorphism between different isolates and more than one viral genotype per isolate. There was a clear correlation between the appearance of clinical symptoms in HIV infection and high levels of viral replication.

Blotting, Southern↗
PubMed1990

Detection of hepatitis B virus DNA in serum by polymerase chain reaction amplification and microtiter sandwich hybridization.

We have developed a microtiter sandwich hybridization assay for the detection of polymerase chain reaction (PCR)-amplified hepatitis B virus (HBV) sequences. This assay utilizes an enzyme-linked immunosorbent assay-like format in which cloned DNA containing a sequence complementary to half of one PCR product strand is immobilized in microtiter wells. A biotin-labeled DNA sequence complementary to the other portion of the same PCR product strand is used as the probe. The DNAs from 69 hepatitis B surface antigen-positive serum samples and 16 antigen-negative control samples were amplified by the PCR procedure, and the product was detected by Southern and sandwich hybridization. Both detection procedures were capable of detecting as few as five copies of HBV DNA. Compared with Southern hybridization, the sandwich hybridization assay exhibited a sensitivity of 100% and a specificity of 95% for the detection of amplified HBV sequences. Unlike Southern hybridization, however, the sandwich hybridization assay employs a nonradioactive probe and allows easy handling of large numbers of samples. DNA was detected in 74% of the antigen-positive samples. All of the antigen-negative samples (healthy blood donors) were negative for HBV DNA by both procedures.

Base Sequence↗
PubMed1989

Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis.

We measured antibody (anti-HCV) to hepatitis C virus, which causes non-A, non-B hepatitis, by radioimmunoassay in prospectively followed transfusion recipients and their donors. Of 15 patients with chronic non-A, non-B hepatitis documented by liver biopsy, all seroconverted for the antibody; of 5 with acute resolving non-A, non-B hepatitis, 3 (60 percent) seroconverted. The development of anti-HCV was delayed (mean delay, 21.9 weeks after transfusion, or 15 weeks after the onset of clinical hepatitis) and took approximately one year in one patient. Antibody has persisted in 14 of the 15 patients with chronic disease (mean follow-up, greater than or equal to 6.9 years; maximum, greater than or equal to 12), but has disappeared in the 3 with acute resolving disease after a mean of 4.1 years. Anti-HCV was detected in samples of donor serum given to 14 (88 percent) of the 16 anti-HCV-positive patients for whom all donor samples were available. Only 33 percent of the anti-HCV-positive donors tested had an elevated serum concentration of alanine aminotransferase; 54 percent were positive for antibody to the hepatitis B core antigen (anti-HBc). We conclude that hepatitis C virus is the predominant agent of transfusion-associated non-A, non-B hepatitis and that screening of donors for anti-HCV could prevent the majority of cases of the disease. "Surrogate" assays for anti-HBc and alanine aminotransferase would have detected approximately half the anti-HCV-positive donors involved in the transmission of hepatitis that we identified.

Acute Disease↗
PubMed1989

What do western blot indeterminate patterns for human immunodeficiency virus mean in EIA-negative blood donors?

To investigate the specificity of western blot indeterminate (WBi) patterns for antibodies to the human immunodeficiency virus type 1 (HIV-1), 100 enzyme immunoassay (EIA) negative donors from whom prospectively obtained recipient sera were available were tested by WB. 20 were WBi, with p24 being the predominant (70%) and generally the only band. Among recipients of WBi blood, 36% were WBi in their 6 month post-transfusion sample, but so were 42% of a control population that had received only WB-negative blood. When serial samples from recipients with a WBi pattern were tested on two occasions, only 35% of results were reproducible. No reciepient of WBi blood became EIA positive, true positive for WB, positive for HIV-1 antigen, or positive for EIA reactivity against recombinant p24 or gp41. The polymerase chain reaction was negative for gag and env HIV-1 sequences in all donors and recipients. Thus WBi patterns are exceedingly common in randomly selected donors and recipients and such patterns do not correlate with the presence of HIV-1 or the transmission of HIV-1 from donor to recipient.

Blood Donors↗
PubMed1989

Clinical implications of positive tests for antibodies to human immunodeficiency virus type 1 in asymptomatic blood donors.

Of 693,000 volunteer blood donors in Washington, D.C., who were screened for infection with human immunodeficiency virus type 1 (HIV-1) from July 1985 through December 1988, 284 tested positive on both enzyme immunoassay and Western blot assay. To determine the clinical importance of confirmed positive test results in asymptomatic blood donors, we followed 156 donors with positive Western blot assays and 80 donors with positive enzyme immunoassays but negative or indeterminate Western blots at 6-month intervals for a mean of 28 months. As compared with Western blot-negative persons, those with positive Western blots were significantly more likely to be black, male, and first-time donors and to have a history of venereal disease, generalized lymphadenopathy on examination, CD4-cell counts lower than 0.4 x 10(9) per liter, IgG levels higher than 18 g per liter, and antibody to hepatitis B core antigen on initial evaluation. In 17 (11 percent) of the Western blot-positive donors, the disease progressed to Class IV (symptomatic disease), according to the Centers for Disease Control system. CD4 counts below 0.2 x 10(9) per liter, IgA levels above 4 g per liter, abnormal proliferative responses to tetanus toxoid, and positive viral cultures were the strongest predictors of disease progression. Among the 80 donors with repeatedly reactive assay results but either negative or indeterminate Western blot assays, there was no evidence of HIV exposure in their histories, physical examinations, or laboratory evaluations, and manifestations of HIV infection developed in none of them. We conclude that a small number of persons with HIV infection continue to donate blood, despite attempts to exclude them, but that donors who test positive on enzyme immunoassay but persistently negative or indeterminate on Western blot assay probably do not represent a risk for the transmission of HIV.

AIDS-Related Complex↗
PubMed1989

Development of a direct-binding enzyme-linked immunosorbent assay as an alternate assay for anti-hepatitis B core.

Anti-hepatitis B core (anti-HBc) screening is now used in some countries as a surrogate test to reduce the incidence of posttransfusion non-A, non-B hepatitis. The purpose of this study was to develop and standardize an alternate assay, for the detection of anti-HBc, based on a direct-binding enzyme-linked immunosorbent assay (db-ELISA). Microtiter plates were coated with recombinant DNA hepatitis B core antigen. Patient antibody was detected spectrophotometrically using a goat, anti-human immunoglobulin conjugated with horseradish peroxidase. The db-ELISA was compared to a standard competitive-binding ELISA (cb-ELISA). A competitive-binding radioimmunoassay (cb-RIA) was used as the reference assay for this study. The specificity of the cb-ELISA was 50% and the positive predictive value was 64% when compared to the cb-RIA. The specificity and positive predictive value of the db-ELISA were both 97% when compared to the cb-RIA. Based on titration studies, the sensitivity of the db-ELISA is at least equal to the cb-RIA. Based on the data generated in this study, the db-ELISA appears to be an acceptable screening test for anti-HBc.

Enzyme-Linked Immunosorbent Assay↗
PubMed1989

A novel virus-like infectious agent in patients with AIDS.

A novel virus-like infectious agent (VLIA), obtained by direct transfection of DNA from Kaposi's sarcoma of a patient with acquired immune deficiency syndrome (AIDS), was transmissible from culture to culture by cell-free filtrate. VLIA contained an outer limiting membrane and had a buoyant density of 1.17-1.20 g/ml in a sucrose gradient. The DNA genome of VLIA was estimated to be greater than 150 kilobase (kb) pairs and carried repetitive sequences. An 8.6 kb pair cloned probe (psb-8.6) and a 2.2 kb pair cloned probe (psb-2.2) of VLIA detected specific sequences in DNA of VLIA infected cells, but not in DNA of uninfected NIH/3T3 cells. By Southern blot hybridization analysis, VLIA was distinct from all known members of human herpes virus, from vaccinia virus, monkey herpes virus saimiri (HVS), and mouse cytomegalovirus (MCMV). Using synthetic primers with the VLIA specific DNA sequences and the polymerase chain reaction (PCR) method, we detected VLIA sequences in DNA isolated from 7 out of 10 patients with AIDS. VLIA infection was identified in spleen, liver, brain, lymph node, Kaposi's sarcoma tissues, or peripheral blood mononuclear cells from these patients, but not in 5 different organs and a tumor from 5 subjects without AIDS. Antiserum raised against VLIA in rabbit positively immunostained brain and lymph node tissues from these AIDS patients.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Fatal infection of silvered leaf monkeys with a virus-like infectious agent (VLIA) derived from a patient with AIDS.

Four silvered leaf monkeys, inoculated with a virus-like infectious agent (VLIA) derived from transformed NIH/3T3 cells (sb51) transfected with Kaposi's sarcoma DNA of an AIDS patient, showed wasting syndromes and died in 7-9 months. Two monkeys had a transient lymphadenopathy in earlier stages. Two moribund animals showed lymphopenia. Although 3 of the VLIA inoculated monkeys had persistent low grade fever early in the infection, the animals became afebrile in the later stages. One VLIA inoculated animal had a prominent antibody response, which occurred 7 months after VLIA inoculation. The other 3 monkeys had a transient or poor antibody response in the later stages. These 3 animals revealed periodic VLIA antigenemia during the course of the experiment. A control monkey was killed 8 months after the last VLIA inoculated monkey succumbed and showed neither an antibody response nor evidence of antigenemia. VLIA-specific DNA could be directly detected in necropsy tissues of all 4 monkeys inoculated with VLIA using the polymerase chain reaction method. VLIA infection was identified in all 4 spleens, 2 of 4 livers, 1 of 2 kidneys, and all 3 brains tested from these 4 animals, but not in the tissues from the control monkey. The necropsy examination of the 4 VLIA inoculated animals revealed no opportunistic infections, acute inflammatory lesions, malignancy or cause of death other than VLIA infection. We believe that the VLIA caused a fatal systemic infection in these monkeys.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Virus-like infectious agent (VLIA) is a novel pathogenic mycoplasma: Mycoplasma incognitus.

The newly recognized pathogenic virus-like infectious agent (VLIA), originally reported in patients with AIDS but also known to be pathogenic in previously healthy non-AIDS patients and in non-human primates, was cultured in cell-free conditions using a modified SP-4 medium and classified as a member of the order Mycoplasmatales, class Mollicutes. The infectious microorganism is tentatively referred to as Mycoplasma incognitus. M. incognitus has the unique biochemical properties of utilizing glucose both aerobically and anaerobically, as well as having the ability to metabolize arginine. Among all known human mycoplasmas, these specific biochemical characteristics were found previously only in a rarely isolated species, M. fermentans. In comparison with M. fermentans, M. incognitus appears to be even more fastidious in cultivation requirements and fails to grow in all tested mycoplasma media other than modified SP-4 medium. In addition, M. incognitus grows much more slowly, has a smaller spherical particle size and occasional filamentous morphology, and forms only irregular and very small colonies with diffuse edges on agar plates. Antigenic analysis using polyclonal and monoclonal antibodies and DNA analysis of sequence homology and restriction enzyme mappings in M. incognitus, M. orale, M. hyorhinis, M. hominis, M. pneumoniae, M. fermentans, M. arginini, M. genitalium, M. salivarium, Ureaplasma urealyticum, and Acholeplasma laidlawii revealed that M. incognitus is distinct from other mycoplasmas, but is most closely related to M. fermentans.

Animals↗
PubMed1989

Identification of Mycoplasma incognitus infection in patients with AIDS: an immunohistochemical, in situ hybridization and ultrastructural study.

Monoclonal antibodies (Mabs) were developed against antigens from a pure culture of Mycoplasma incognitus grown in modified SP-4 medium. All the Mabs obtained were shown to react only with M. incognitus, and not with other species of human mycoplasma. The Mabs identified M. incognitus immunohistologically in thymus, liver, spleen, lymph node, or brain from 22 patients with AIDS, as well as in 2 placentas delivered by patients with AIDS. Using an 35S-labeled DNA probe specific for M. incognitus and in situ hybridization technique, we also identified M. incognitus-specific genetic material in these tissues. Furthermore, ultrastructural studies of the specific areas of tissues which were highly positive for M. incognitus antigens revealed characteristic structures of mycoplasma organisms. These mycoplasma-like particles could be identified intracellularly and extracellularly. Histopathology of the tissues infected by M. incognitus varied from no pathological changes to fulminant necrosis with or without an associated inflammatory reaction. M. incognitus, a novel pathogenic mycoplasma, was cytopathic and cytocidal.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Comparison of four different antigen-capture assays for the detection of human immunodeficiency virus antigen (HIV-Ag).

Three enzyme immunoassays (EIA), two polyclonal and one monoclonal, and one polyclonal radioimmunoassay (RIA) for the detection of human immunodeficiency virus antigen (HIV-Ag) have been evaluated and compared. The three EIAs had similar sensitivity in detecting HIV viral lysate and were more sensitive than the RIA, which was the only assay with a one-step probing-detection format. However, the EIA that used a monoclonal anti-p24 as the capturing reagent was unable to detect any of the serial supernatant samples of a positive viral culture from an HIV-infected patient. Only the two polyclonal, non-p24-restricted assays were able to detect an unusual expression of HIV-Ag in the serum of an acute HIV-infected patient. Overall, the sensitivity of HIV-Ag capture assays was enhanced when (a) the capture antibody was polyclonal rather than monoclonal, (b) the polyclonal antibody was broad rather than p24-restricted, and (c) the probing-detecting procedure was in a two-step format rather than one-step format. In addition, the use of neutralizing assays to confirm the results was absolutely necessary.

Antibodies, Monoclonal↗