Biomedical subjects
Publications and source records attributed to D M Yajko.
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A mycobacterial DNA probe (designated X) was recently developed to help identify Mycobacterium avium complex (MAC) isolates that are nonreactive with probes specific for M. avium or Mycobacterium intracellulare. The prevalence of X probe-positive mycobacteria in clinical specimens and their role in causing disease is unknown. Using a DNA probe kit that includes the X probe, we characterized 100 consecutive clinical MAC isolates as M. avium, M. intracellulare, or X. Lysates from 81 of the isolates reacted with the M. avium probe, 13 with the M. intracellulare probe, 3 with the X probe, and 3 failed to hybridize with any of the probes. All three X-positive isolates were recovered from sputa of patients who were recent immigrants to the United States and who presented with hemoptysis. One isolate was from a Hispanic man infected with human immunodeficiency virus type 1 (HIV-1) and the other 2 were from Filipino patients with no HIV-1 risk factors. This study also showed a higher than expected number of M. intracellulare isolates from blood and cerebrospinal fluid of HIV-1-infected patients.
The intracellular activities of clarithromycin and erythromycin, alone and in combination with other antimicrobial agents, were tested against Mycobacterium avium complex (MAC) strains inside mouse J774 cells and inside alveolar macrophages obtained from human immunodeficiency type 1-infected individuals. Clarithromycin alone had greater intracellular activity than erythromycin alone, and drug combinations that included clarithromycin were usually more active than combinations that included erythromycin.
Bacteriophage T4 DNA was detected and analyzed inside E. coli by dual-laser flow cytometry using a dye combination of Hoechst 33258 (H33258) and chromomycin A3 (CA3) which bind to A-T- and G-C-rich regions of DNA, respectively. An exponentially-growing culture of E. coli ATCC 11303 was infected with T4 bacteriophage at a 1:1 multiplicity of infection. Samples were taken immediately and at 5 min intervals and placed on ice to interrupt viral replication. The samples were then centrifuged, ethanol-fixed, stained with H33258 and CA3, and analyzed by flow cytometry. Twenty-five minutes post-infection, a population of cells which contained T4 DNA began to appear on both a bivariate contour plot and a frequency histogram plot of the data. By 35 min, T4 DNA-containing cells could be distinguished from E. coli cells containing little or no T4 DNA. The ratio of CA3:H33258 fluorescence was then used to calculate the % G + C value for T4 DNA inside E. coli. A value of 35.6 +/- 0.2% was obtained, which agrees with % G + C values determined by traditional methods. These results demonstrate that dual-laser flow cytometry can be used to study viral DNA inside the bacterial host.
This study sought to better characterize the natural history of AIDS-associated disseminated Mycobacterium avium complex (MAC) infection. Towards that end two retrospective studies were done: a case-control survival study and a MAC respiratory colonization study. Among 137 consecutive patients who had a sterile body site cultured for mycobacteria within 3 months of their first AIDS-defining episode of Pneumocystis carinii pneumonia, median survival was significantly shorter in those with disseminated MAC infection (107 days; 95% confidence interval [CI] 55-179) than those with negative cultures (275 days; 95% CI 230-318; P less than .01), even after controlling for age, absolute lymphocyte count, and hemoglobin concentration. Among 34 patients with AIDS and respiratory MAC colonization, 22 later developed disseminated infection (65% predictive value for subsequent MAC dissemination). Disseminated MAC infection was associated with significantly shorter survival for patients with AIDS, and the presence of MAC in respiratory specimens has substantial predictive value for subsequent disseminated infection.
Measurements of the activities of antimicrobial agents against the Mycobacterium avium complex (MAC) usually do not take into consideration the intracellular location of the organism. A recent study using mouse macrophage continuous cell line J774 (D. M. Yajko, P.S. Nassos, C. A. Sanders, and W. K. Hadley, Am. Rev. Respir. Dis. 140: 1198-1203, 1989) showed that certain combinations of antimicrobial agents are able to kill MAC inside macrophages and suggested that the J774 cell line could be used as a model for screening of drugs for intracellular activity against MAC. As a test of the validity of this model, alveolar macrophages were isolated from the bronchoalveolar lavages of 36 patients who had AIDS or an AIDS-related condition or were considered to be at risk for AIDS. The macrophages were infected with MAC and then treated with a drug or drug combination for 48 to 72 h. Survival of MAC was measured over time in drug-treated macrophages and untreated control macrophages. No single drug or two-drug combination that was tested was able to cause a decrease in the survival of every one of the MAC strains used in the study. However, several three-drug combinations that had been shown to cause a decrease in survival of all MAC strains inside J774 cells also caused a decrease in survival of all MAC strains inside alveolar macrophages from patients. The good agreement between these results and those obtained previously with J774 cells gives further evidence of the usefulness of the simpler J774 model for screening of drugs for intracellular activity against MAC.
Over the past several years there has been a large increase in the recovery of Mycobacterium avium complex (MAC) isolates from respiratory specimens submitted to the clinical laboratory at San Francisco General Hospital (SFGH). This increase in MAC recovery correlates with an increase in the number of cases of acquired immunodeficiency syndrome (AIDS) in the community. Although it is well known that MAC is often isolated from patients with AIDS, the isolation of MAC from respiratory specimens is often attributed to contamination of the specimen with MAC organisms present in the environment. To determine whether the increase in MAC isolates recovered at SFGH was due to an increase in environmental contamination of specimens or to the increase in our AIDS patient population, we conducted a study of the prevalence of MAC in respiratory specimens from AIDS versus non-AIDS patients. Results of specimens submitted to the clinical laboratory at SFGH for culture of mycobacteria were reviewed over a 12-yr period, from 1977 through 1988. The prevalence of MAC in respiratory specimens from AIDS and non-AIDS patients was determined for 4 yr during this period: the pre-AIDS year 1977; the first year AIDS was reported in San Francisco, 1981; 1984; and 1987. In 1977 and 1981 the prevalence of MAC in respiratory specimens was less than or equal to 0.5%, and all MAC isolates were recovered from non-AIDS patients. In 1984 the prevalence of MAC in respiratory specimens for AIDS and non-AIDS patients was 6.5 and 0.3%, respectively, and in 1987, 8.8 and 0.3%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
A collaborative study was undertaken at two institutions to assess the performance of a direct fluorescent-antibody stain for the detection of Pneumocystis carinii in respiratory specimens from patients with known or suspected human immunodeficiency virus type 1 infections. A total of 163 specimens (125 induced sputa, 37 bronchoalveolar lavage fluids, and 1 tracheal aspirate) from 124 patients were examined by using modified Giemsa (Diff-Quik; Baxter American Scientific Products, Chicago, Ill.) and direct fluorescent-antibody stains. A total of 73 specimens contained P. carinii, which was detected in 66 (92%) of the specimens by using the modified Giemsa and in 71 (97%) of the specimens by using the fluorescent-antibody stain. One bronchoalveolar lavage fluid specimen in which P. carinii was detected only with the fluorescent-antibody stain was determined to be a false-positive based on subsequent clinical evaluation of the patient. Although the overall time for processing and examining specimens stained with either stain was not significantly different for those specimens containing P. carinii, considerably less time was required for microscopic examination of those fluorescent-antibody-stained specimens lacking P. carinii.
Two prospective studies were undertaken to evaluate a commercial indirect fluorescent-antibody (IFA) stain for the detection of Pneumocystis carinii in respiratory specimens from individuals at risk for or with the acquired immunodeficiency syndrome. The first study compared IFA with Diff-Quik (DQ; a rapid Giemsa-like stain) for detecting P. carinii in 95 induced sputa obtained from 77 asymptomatic patients who had survived one previous episode of P. carinii pneumonia and who were being treated prophylactically with aerosolized pentamidine. Only one induced sputum specimen was found to contain P. carinii; organisms were detected by both stains. The second study compared the performance of the IFA stain versus DQ, modified toluidine blue O, and Gomori methenamine silver stains for detecting P. carinii in symptomatic individuals at risk for or with acquired immunodeficiency syndrome. Of 182 specimens examined, P. carinii was detected in 105 by one or more stains; the DQ stain detected 73 (70%), the modified toluidine blue O stain detected 75 (71%), the Gomori methenamine silver stain detected 76 (72%), and the IFA stain detected 95 (90%). The IFA stain was more sensitive (P less than 0.01) than the other traditional stains for detecting P. carinii; however, a subsequent clinical evaluation revealed that a subset of IFA-positive-only specimens were from patients whose clinical symptoms resolved without specific anti-P. carinii therapy.
Cyclic AMP levels doubled in Myxococcus xanthus under conditions in which cells aggregate and form fruiting bodies. In liquid medium, glycerol- or dimethyl sulfoxide-induced sporulating cultures exhibited a sharp but transient rise in cyclic AMP concentration after 45 min.
We studied mutants of E. coli originally identified as being deficient in either endonuclease II (deoxyribonucleate oligonucleotidohydrolase, EC 3.1.4.30) or exonuclease III [deoxyribonucleate (double-stranded) 5'-nucleotidohydrolase, EC 3.1.4.27] activity. Twelve independently derived mutants were tested, including three new endonuclease II mutants. Deficiency of one enzyme was always accompanied by deficiency of the other. Furthermore, temperature-sensitivity of one activity was always accompanied by temperature-sensitivity of the other, and the enzymes were co-purified. The results suggested a physical association between exonuclease III and endonuclease II, which may be of advantage in the excision-repair of DNA. A thermolabile endonuclease II was purified from one of the new mutants, indicating that it had an altered structural gene. This mutation, and all similar ones mapped by genetic transduction, was located between the pncA and aroD genes on the E. coli chromosome. One mutant had a prolonged generation time, an increased sensitivity to the alkylating agents methyl-methanesulfonate and mitomycin C, and a decreased plating efficiency for bacteriophage lambda, but no marked sensitivity to ultraviolet or gamma-irradiation. Its enzymatic and biological abnormalities were simultaneously revertible, suggesting they were caused by a single mutation. These results suggested a role for these enzymes in normal cell growth processes and in the repair of alkylation damage.