Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0021051 (immunodeficiency)
 71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transmission of multidrug-resistant strains of Mycobacterium tuberculosis (MDR-TB) presents a serious problem for infection control in hospitals, particularly in the context of co-infection with the human immunodeficiency virus (HIV). We report on the use of molecular genetic tools to allow rapid assessment of samples from patients potentially infected with MDR-TB. Sputum and bronchoalveolar lavage samples were obtained from two HIV-positive patients with suspected tuberculosis, who had previous contact with a known MDR-TB index case. Polymerase chain reaction (PCR) was used directly on clinical samples to amplify genetic loci associated with rifampicin resistance (rpoB), and strain-specific polymorphisms (the direct repeat (DR) region). Drug resistance was determined using a commercially available kit for detection of point mutations in the rpoB gene (Inno-Lipa RifTB; Innogenetics, Belgium), and confirmed by nucleotide sequencing. Strain variation was determined using the spoligotyping method, based on the presence or absence of variable linker sequences within the DR region. In one patient, infection with a MDR strain identical to that of a known index case was demonstrated. A second patient, although positive for M. tuberculosis, was found to be infected with a rifampicin-sensitive strain. Results were obtained within 48 h, allowing appropriate treatment to be initiated and infection control measures to be implemented. PCR-based tests for strain-typing and for identification of rifampicin resistance provide important tools for identifying patients with MDR-TB and for rapid monitoring of potential nosocomial spread of MDR-TB. Prompt confirmation or exclusion of possible transmission allows early clinical intervention to prevent future outbreaks of multidrug-resistant M. tuberculosis.
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PMID:Rapid detection of multidrug-resistant tuberculosis. 916 56

The method of the poly A-linked colorimetric reverse transcriptase assay (PAC-RTA) was developed and evaluated for the measurement of Mg(2+)-dependent reverse transcriptase (RT) activity of feline immunodeficiency virus (FIV). PAC-RTA was first evaluated for the detection of RT activity in the culture supernatant of FIV Petaluma strain. The detection limit of RT activity by PAC-RTA was about 10-fold better than that by the conventional non-radioisotopic RT assay kit. Then, PAC-RTA was evaluated for the indication of FIV isolation from cats naturally infected with FIV. FIV was isolated from peripheral blood mononuclear cells of 9 FIV-seropositive cats. The time course appearance of RT activity measured by PAC-RTA corresponded with the analysis of FIV antigen expression by indirect immunofluorescence. Finally, PAC-RTA evaluated the drug susceptibility of FIV. MYA-1 cells (feline T-lymphoblastoid cells) were infected with FIV and were cultured in the presence of various concentrations of anti-human immunodeficiency virus agents such as azidothymidine (AZT) or dextran sulfate. An inverse relationship between the RT activities and the concentrations of these agents in the culture supernatant was confirmed by PAC-RTA. PAC-RTA is easy to perform without using radioactive materials, and one plate can handle 96 samples at one time. By monitoring the RT activity, this assay is a useful method for FIV studies such as viral replication and drug susceptibility.
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PMID:Measurement of reverse transcriptase of feline immunodeficiency virus by poly A-linked colorimetric assay. 923 15

We report on the development of a rapid nested PCR protocol for the detection of Pneumocystis carinii DNA in bronchoalveolar lavage (BAL) specimens in which the protocol included the use of a commercially available DNA extraction kit (GeneReleaser). GeneReleaser enabled us to obtain amplification-ready DNA within 20 min without requiring the purification of the DNA. The nested PCR was performed with the primers pAZ102-E, pAZ102-H, and pAZ102-L2 (A. E. Wakefield, F. J. Pixley, S. Banerji, K. Sinclair, R. F. Miller, E. R. Moxon, and J. M. Hopkin, Lancet 336:451-453, 1990.). Results were obtained in about 4 h with the adoption of denaturation, annealing, and extension steps shortened to 20 seconds. The sensitivity of the nested PCR was tested with a P. carinii cyst suspension and was found to be less than one cyst (one to eight nuclei). The detection limit was the same with the use of GeneReleaser or proteinase K-phenol chloroform for DNA extraction. The nested PCR assay was prospectively compared with staining with Giemsa and methenamine silver stains for the detection of P. carinii in 127 BAL samples from 105 human immunodeficiency virus-infected patients investigated for acute respiratory illness. Twenty-five BAL specimens (20%) were positive by staining and the nested PCR and 25 (20%) were negative by staining and positive by the nested PCR. These 25 BAL specimens with conflicting results were obtained from 23 patients, 82% of whom were receiving prophylactic therapy against P. carinii pneumonia (PCP). Only two patients were diagnosed with possible PCP. The final diagnosis was not PCP for 20 patients who were considered to be colonized or to have a low level of infection. This colonization is not of clinical importance but is of epidemiological importance. Our rapid, simple, and sensitive amplification protocol may be performed in clinical laboratories for the routine diagnosis of PCP with BAL specimens.
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PMID:Rapid detection of Pneumocystis carinii in bronchoalveolar lavage specimens from human immunodeficiency virus-infected patients: use of a simple DNA extraction procedure and nested PCR. 935 Jul 26

Over a 4-year period, the Roche Amplicor kit was used in a United Kingdom reference laboratory for the detection or confirmation of human immunodeficiency virus (HIV) type 1 infection, particularly in infants born to HIV-infected mothers. Of 408 specimens from adults and older children tested, the 122 seronegative specimens were all Amplicor negative. Of the 286 seropositive specimens, 268 were Amplicor positive. On the basis of these results, the Amplicor assay has a specificity of 100% and a sensitivity of 93.7%. In addition, for 247 specimens from infants and young children, serological results may not have been diagnostic because of placental transfer of maternal antibodies. Forty-eight were Amplicor positive, and of the 199 Amplicor-negative specimens, 19 were assumed to be false negative on the basis of clinical data, serological markers (including p24 antigen), and/or results for previous or follow-up specimens. This represents a sensitivity of 75% for the Amplicor test for specimens from patients under 2 years of age. Of these 37 false-negative specimens plus 2 specimens from other laboratories, 31 could be characterized by amplifying extracted material from them by an in-house nested gag PCR spanning the Amplicor target region. The amplicons were sequenced and found to represent subtypes A (35.5%), B (22.6%), C (22.6%), D (16.1%), and G (3.2%). False-negative results by the Amplicor assay may be ascribed to low-target copy number, the physical behavior of one primer (SK462), and sequence variation in the target region of the other primer (SK431).
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PMID:Performance of the Amplicor human immunodeficiency virus type 1 PCR and analysis of specimens with false-negative results. 935 Jul 45

We investigated the effects of conditions often encountered during handling, transit, and storage of blood specimens on the quantity of detectable human immunodeficiency virus (HIV) RNA in plasma. HIV RNA copy numbers were measured with a commercially available assay (the Amplicor HIV-1 Monitor test kit). Variables examined were the time to processing of blood and plasma, the holding temperature of blood and plasma prior to processing, the effect of freezing and thawing of plasma, and the use of different anticoagulants. The relationship between the HIV RNA copy number and the HIV isolation rate by peripheral blood mononuclear cell (PBMC) coculture was also examined. We found that RNA copy numbers were maintained to within 0.5 log10 (approximately threefold) in blood and plasma samples held at room temperature or 4 degrees C for up to 3 days and remained stable despite (limited) freezing and thawing of the plasma. HIV RNA copy numbers were also maintained after long-term storage of plasma at -70 degrees C. The ability to isolate HIV from PBMCs was directly proportional to the HIV RNA copy number.
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PMID:Stability of human immunodeficiency virus RNA in blood specimens as measured by a commercial PCR-based assay. 946 65

Enzyme immunoassay (EIA) is an ideal method for screening large numbers of patients for syphilis. We evaluated a novel immune-capture EIA (ICE Syphilis; Murex Diagnostics) that uses three recombinant Treponema pallidum antigens (TpN15, TpN17, and TpN47) and compared the results with those obtained by the native T. pallidum antigen EIA (Captia SelectSyph-G; Centocor) that we currently use for the serodiagnosis of syphilis. Specificity was evaluated by screening 1,184 unselected serum specimens in parallel by the ICE Syphilis and SelectSyph-G assays, while sensitivity was tested with a panel of 101 serum specimens containing antitreponemal antibodies (treated and untreated) from patients with various stages of infection. The specificity of the ICE Syphilis EIA (99.8%) on screening was significantly higher (P < 0.02) than that of the SelectSyph-G EIA (99.2%). The sensitivity of the ICE Syphilis EIA was significantly higher (P < 0.01) than that of the SelectSyph-G EIA on both initial (99 versus 91.4%) and repeat (100 versus 92.4%) testing. The ICE Syphilis EIA was also significantly more sensitive (P < 0.01) than the fluorescent treponemal antibody-abs (92.4%) but not the T. pallidum hemagglutination assay (97.1%). Sera containing antitreponemal antibodies gave a much higher antibody index (absorbance of test serum/kit cutoff) by the ICE Syphilis EIA than by the SelectSyph-G EIA. This combined with the overall high sensitivity makes the ICE Syphilis EIA an ideal test for excluding or detecting treponemal infection in human immunodeficiency virus (HIV)-infected patients. The ICE Syphilis EIA was positive with sera from all 15 HIV-infected patients in the study, whereas sera from 3 HIV-infected patients were negative by the SelectSyph-G EIA. We conclude that the high sensitivity and specificity of the ICE Syphilis EIA and its suitability for automation make it an ideal screening test.
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PMID:Novel recombinant-antigen enzyme immunoassay for serological diagnosis of syphilis. 954 8

Procedures for quality control (QC) in a laboratory that concentrates on cytokine and soluble marker measurements in biological fluids are outlined. Intra-assay, interassay, and interlaboratory experiences are presented. Plasma and serum beta2-microglobulin (beta2M) and neopterin test data are presented in greatest detail, along with substantial tumor necrosis factor alpha (TNF-alpha), gamma interferon, soluble interleukin-2 receptor-alpha (sIL-2Ralpha), sTNF-RII, IL-4, and IL-6 data. Recommended QC procedures for cytokine and soluble-marker testing include replicate testing of two or more reference samples provided by the kit manufacturer, replicate testing of in-house frozen reference QC samples that represent normal and abnormal analyte contents, retesting 15 to 20% of randomly selected samples, and comparing normal reference ranges each year. Also, eight cytokines and soluble markers were evaluated in human immunodeficiency virus (HIV)-seronegative and HIV-seropositive individuals stratified on the basis of CD4 T-cell numbers. Levels of some but not all cytokines in serum increased in HIV infection. There was a tendency for cytokines to increase with more advanced disease, defined by reduced CD4 T-cell numbers. Cytokine changes did not relate closely to CD4 level, indicating that separate information was provided by the measurements of TNF-alpha, sTNF-RII, sIL-2Ralpha, beta2M, and neopterin. Serum IL-4 and TNF-alpha levels were not increased. The quality of laboratory data can impact on clinical relevance. Interlaboratory comparisons revealed substantial differences at some sites and documented the need for external proficiency-testing quality assurance programs.
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PMID:Levels of cytokines and immune activation markers in plasma in human immunodeficiency virus infection: quality control procedures. 980 30

The detection of Pneumocystis carinii DNA in blood by PCR could be useful for studying the natural history of pneumocystosis and could also be a noninvasive diagnostic method. The results of previous studies are nevertheless conflicting. In our study, we compared three commercially available DNA extraction kits (GeneReleaser, QIAamp Tissue Kit, and ReadyAmp Genomic DNA Purification System) and proteinase K and proteinase K-phenol-chloroform treatments for the extraction of P. carinii DNA from dilutions of a P. carinii f. sp. hominis cyst suspension mixed with human whole blood. A rapid and simple nested PCR protocol which amplifies a portion of the mitochondrial large-subunit rRNA gene was applied to all the extraction products. The QIAmp Tissue Kit was the most effective kit for the isolation of amplification-ready P. carinii DNA and was used with nested PCR for the testing of whole-blood specimens from 35 immunocompetent control patients and 84 human immunodeficiency virus (HIV)-infected patients investigated for pulmonary disease and/or fever. In HIV-infected patients, P. carinii DNA was detected by nested PCR in blood samples from 3 of 14 patients with microscopically proven P. carinii pneumonia, 7 of 22 patients who were considered to be colonized with P. carinii, and 9 of 48 patients who were neither infected nor colonized with P. carinii. P. carinii DNA was not detected in blood specimens from the 35 immunocompetent patients. P. carinii DNA in blood might represent viable P. carinii organisms or DNA complexes released from pulmonary phagocytes. In conclusion, P. carinii DNA may be detected in whole blood from HIV-infected patients, but the nature and the meaning of the circulating form of P. carinii remain to be established.
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PMID:Detection of Pneumocystis carinii DNA in blood specimens from human immunodeficiency virus-infected patients by nested PCR. 985 76

The effect of 44 different metal ions (Ag+, Al3+, As(O-)2, Au3+, Ba2+, Be2+, Bi3+, Cd2+, Ce3+, CO2+, Cr(O2-)4, Cr3+, Cs+, Cu2+, Fe3+, Fe2+, Ga3+, Ge4+, Hg2+, Ir4+, La3+, Li+, Mn2+, MO6+, Ni2+, OS4+, Pb2+, Pt4+, Rb+, Rh3+, Sb5+, Se(O2-)4, Se(O2-)3, Sn2+, Sr2+, Th4+, T1+, U(O2+)2, V(O-)3, VO2+, W(O2-)4, Y3+, Zn2+, and Zr4+) on the activity of the reverse transcriptase (RT) of the human immunodeficiency virus (HIV-1) was investigated in vitro. For this study, the RT activity assay was carried out by means of an enzyme-linked immunosorbent assay (ELISA) kit, using the template/primer hybrid poly(A) oligo(dT)15, which required some modifications: (1) possible interfering metal chelators (such as EDTA) in the original lysis buffer were avoided, and a new buffer (50 mM Tris-NO3, pH 7.8) was used throughout; (2) an amount of 2 ng of RT per well was considered to be optimal after checking the linearity of the reaction with increasing amounts of enzyme; (3) an incubation temperature of 37 degrees C and an incubation time of 1 h were chosen after preliminary studies in a wide range of temperature and time. At an incubation temperature > or = 40 degrees C, there was a dramatic loss of enzymatic activity. In addition, when RT alone was preincubated for 1 h at 5 degrees C, 25 degrees C, and 37 degrees C, there was a large (83%) loss of activity at 37 C as compared to that at 5 degrees C. These results are indicative of enzyme thermolability, which is higher in the absence of substrates. The effect of metal ions on RT activity was tested using two different metal salt concentrations (10(-4) M and 10(-5) M). Under such experimental conditions, the presence of five metal ions (Pt4+, Ag+, Rh3+, Zn2+, and Hg2+) decreased the RT activity in a dose-response fashion. The observed order of effectiveness with respect to inhibition was Pt4+ > Ag+ > Rh3+ > Zn2+ = Hg2+. Estimated mean inhibitory concentrations (IC50) were 7.8 microM for (NH4)2PtCl6, 14.1 microM for AgNO3, 46.8 microM for RhCl3, 53.7 microM for Zn(SO)4, and 56.2 microM for Hg(NO3)2. Because these data are of the same order of magnitude as the corresponding values related to other RT inhibitors used in anti-AIDS therapy, metal compounds or their derivatives could give an interesting contribution in the development of new RT inhibitors for clinical use.
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PMID:Effects of trace metal compounds on HIV-1 reverse transcriptase: an in vitro study. 1032 22

This study was performed to determine the feasibility of using saliva as a diagnostic medium for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 under nonlaboratory conditions and to evaluate the performance characteristics of such a test. We developed for this purpose a self-contained kit (Saliva. Strip [ST]), which combines the collection and processing, as well as the analysis, of the specimen. The kit's performance was evaluated in a blinded study. Saliva collection was facilitated with a specially designed device that contains a sample adequacy indicator, and immunochromatography test strips were used for the analysis. A total of 1,336 matched serum and saliva specimens (684 reactive and 652 nonreactive specimens) were tested. We tested sera using an enzyme immunoassay (EIA) and a rapid strip test. Sera reactive in one of the assays were also analyzed by Western blotting. Sensitivity and specificity were 99.4 and 99.4%, respectively, for ST, 100 and 99.1%, respectively, for EIA, and 99.7 and 100%, respectively, for the serum strip test. The saliva test performed well when HIV-2-positive sera or a low-titer performance panel (HIV-1) of serum or plasma specimens were diluted (1:2,000) in nonreactive saliva. Because the methodology we present here uses a noninvasively obtained medium, the methodology may be suitable for use in the field where laboratory support and personnel are limited, such as community outreach programs, doctors' offices, surveillance studies, and community hospitals.
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PMID:A simple saliva-based test for detecting antibodies to human immunodeficiency virus. 1039 66


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