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Biomedical subjects

R C Good

Publications and source records attributed to R C Good.

At least 37 records · Page 2Linked to original sources

Immunologic characterization of a 35-kilodalton recombinant antigen of Mycobacterium tuberculosis.

A 35-kilodalton (kDa) recombinant antigen (35-kDa antigen) produced by Escherichia coli JM107 carrying DNA from Mycobacterium tuberculosis was purified and immunologically examined by in vivo and in vitro methods. A monoclonal antibody (2B2) was produced against the 35-kDa antigen. The protein was purified from the insoluble fraction of the recombinant E. coli strain by either affinity chromatography with the 2B2 monoclonal antibody or preparative isoelectric focusing. In enzyme-linked immunosorbent assay and Western blot (immunoblot) analyses, antibody to 2B2 reacted with whole-cell sonic extracts of M. tuberculosis and other slowly growing mycobacteria but not with two rapid growers, M. chelonae and M. fortuitum. An injection series totaling less than 1 mg of purified protein without adjuvant elicited a humoral response in guinea pigs. In one guinea pig, 10 micrograms of purified protein injected intradermally elicited both a humoral and a cell-mediated response. Results of these studies suggest that the 35-kDa antigen is a membrane-associated protein that stimulates both humoral and cell-mediated immune responses and should be evaluated as a vaccine candidate.

Animals↗

Geographic distribution, frequency, and specimen source of Mycobacterium avium complex serotypes isolated from patients with acquired immunodeficiency syndrome.

Isolates of Mycobacterium avium complex from 727 patients with acquired immunodeficiency syndrome (AIDS) were submitted by medical centers across the United States to the Centers for Disease Control for serotyping. We were able to type 630 (87%) of these isolates by our seroagglutination procedure. Almost all typeable isolates were M. avium (serotypes 1 to 6 and 8 to 11). Blood was the major specimen source for both M. avium and the nontypeable isolates. M. intracellulare serotypes made up only 3% of all isolates from AIDS patients, with sputum being the major specimen source. More than 50% of the isolates originated from either New York or California, with serotype 4 being isolated most frequently in New York and serotype 8 appearing most frequently in California. AIDS patients in Los Angeles had a significantly higher isolation frequency for serotype 8 and a significantly lower one for serotype 4 in comparison with patients in either San Francisco or New York City.

Acquired Immunodeficiency Syndrome↗

The modern mycobacteriology laboratory. How it can help the clinician.

The mycobacteriology laboratory provides the information necessary to diagnose mycobacteriosis and to suggest proper patient management. When the definite diagnosis of disease due to M. tuberculosis is made on the basis of the laboratory result, contact follow-up studies should continue and the patient may be considered infectious if the bacilli were isolated from the sputum. A report of another Mycobacterium species suggests that a contact follow-up is not necessary, that the patient need not be isolated, and that a therapeutic regimen should be based on the results of drug-susceptibility tests with the isolate. Techniques of molecular biology are being used in the laboratory to provide the necessary information quickly for patient management. BACTEC has been accepted into most clinical laboratories to speed reporting of results. Other methods such as genetic and immunologic probes are under development. New DNA probes have been marketed by GenProbe for the identification of cultures of M. tuberculosis, M. avium, and M. intracellulare. HPLC and GLC have been used for the identification of cultures based on unique mycolic acid patterns of the species. Immunologic probes may eventually be the most specific and sensitive, but additional development is necessary. Mycobacteriophage typing of M. tuberculosis isolates has been developed as a tool to aid in epidemiologic studies. These and other technologies are essential in the support of programs for the elimination of tuberculosis.

Bacteriological Techniques↗

Prevalence of nontuberculous mycobacteria in water supplies of hemodialysis centers.

Infection of hemodialysis patients with nontuberculous mycobacteria (NTM) has been associated with water used in reprocessing hemodialyzers. This study was conducted to determine the prevalence of NTM and other bacteria in water samples collected over a 13-week period from 115 randomly selected dialysis centers in the United States. Total viable counts were determined by membrane filter assays; increased recovery of NTM was obtained by dosing a portion of each water sample with 1% formaldehyde (HCHO) before filtering. NTM were widely distributed and occurred with a high frequency in water supplies in dialysis centers. NTM were detected in water from 95 centers (83%), and 50% of all samples examined contained NTM. The results of this study support recommendations to use 4% HCHO or a chemical germicidal equivalent for disinfecting dialyzers that are to be reused.

Disinfection↗

Expression of proteins of Mycobacterium tuberculosis in Escherichia coli and potential of recombinant genes and proteins for development of diagnostic reagents.

Recombinant plasmids containing DNA from Mycobacterium tuberculosis were transformed into Escherichia coli, and three colonies were selected by their reactivity with polyclonal antisera to M. tuberculosis. The three recombinant vectors contained DNA inserts of different sizes flanking a common 4.7-kilobase (kb) sequence. Each recombinant produced 35- and 53-kilodalton proteins (35K and 53K proteins, respectively) which were absent in the control E. coli. In Western blotting experiments, both proteins bound several antisera to M. tuberculosis but not antisera to other commonly isolated mycobacteria. Rabbits immunized with the recombinant 35K protein produced antisera which bound to both the 35K and 53K protein bands, a single 35K protein band present in a culture filtrate of M. tuberculosis, and single protein bands with differing molecular weights in whole-cell homogenates from other Mycobacterium spp. An additional recombinant vector containing a 2.2-kb subclone of the 4.7-kb sequence was constructed and, when used as a probe, demonstrated homology with various fragments of chromosomal digests of selected mycobacteria. Reactivity of this probe to Mycobacterium bovis and M. bovis BCG was indistinguishable from reactivity to M. tuberculosis. Immunoglobulin G reactivity to the 35K antigen was detected in antisera from 8 of 20 persons with active tuberculosis, 4 of 18 persons with leprosy, and none of 14 healthy controls. In contrast, reactivity to various proteins in M. tuberculosis culture filtrate was present in 18 of 20 patients with tuberculosis, 16 to 18 patients with leprosy, and 5 of 14 controls. The production of M. tuberculosis proteins by E. coli circumvents many difficulties encountered in the growth and manipulation of M. tuberculosis and may facilitate the development of better diagnostic and immunizing reagents.

Antibodies, Bacterial↗

Selective procedures for detecting femtomole quantities of tuberculostearic acid in serum and cerebrospinal fluid by frequency-pulsed electron capture gas-liquid chromatography.

Conditions are described for the detection of tuberculostearic acid (10-methyloctadecanoate; C18 X CH3) in cerebrospinal fluid and serum of patients with tuberculous meningitis. C18 X CH3 was found in both the cerebrospinal fluid and serum of patients with tuberculous meningitis at concentrations of 25 to 50 fmol (10(-15) mol). The necessary specificity and sensitivity for detection of C18 X CH3 were obtained by extraction under acid conditions with organic solvent, specific functional group esterification with trichloroethanol, cleanup with disposable reverse-phase sorption chromatography columns, analysis on high-resolution polar and nonpolar capillary columns, and detection by a frequency-pulsed electron capture detector. Use of an IBM 9000 computer equipped with CAP software significantly aided comparison between known C18 X CH3 standards and C18 X CH3 in clinical specimens. Scale expansion and attenuation changes were the major contributions obtained by use of the computer. The data indicate that detection of C18 X CH3 by frequency-pulsed electron capture gas-liquid chromatography may be a valuable aid for early detection of tuberculous meningitis.

Chromatography, Gas↗

Activity of selected beta-lactam antibiotics against Mycobacterium leprae.

Twelve beta-lactam antibiotics were tested for activity against Mycobacterium leprae growing in the foot pads of mice. Two cephalosporins (7-aminocephalosporanic acid and cefuroxime) and one cephamycin (cefoxitin) showed significant activity against M. leprae, and one penicillin (mezlocillin) exerted possible growth-promoting activity. These results suggest that particular molecular structures may be required for activity against M. leprae.

Animals↗

Analysis of mycolic acid cleavage products and cellular fatty acids of Mycobacterium species by capillary gas chromatography.

After growth and experimental conditions were established, the mycolic acid cleavage products, constituent fatty acids, and alcohols of representative strains of Mycobacterium tuberculosis, M. smegmatis, M. fortuitum complex, M. kansasii, M. gordonae, and M. avium complex were determined by capillary gas chromatography. Reproducible cleavage of mycolic acid methyl esters to tetracosanoic (24:0) or hexacosanoic (26:0) acid methyl esters was achieved by heating the sample in a high-temperature muffle furnace. The major constituent fatty acids in all species were hexadecanoic (16:0) and octadecenoic (18:1 omega 9-c, oleic) acids. With the exception of M. gordonae, 10-methyloctadecanoic acid was found in all species; moreover, M. gordonae was the only species tested which contained 2-methyltetradecanoic acid. M. kansasii was characterized by the presence of 2,4-dimethyltetradecanoic acid, M. avium complex by 2-eicosanol, and M. tuberculosis by 26:0 mycolic acid cleavage product. The mycolic acid cleavage product in the other five species tested was 24:0. Although a limited number of strains and species were tested, preliminary results indicate that this gas chromatographic method can be used to characterize mycobacterial cultures by their mycolic acid cleavage products and constituent fatty acid and alcohol content.

Alcohols↗

Rapid radiometric methods to detect and differentiate Mycobacterium tuberculosis/M. bovis from other mycobacterial species.

Rapid methods for the differentiation of Mycobacterium tuberculosis/M. bovis (TB complex) from other mycobacteria (MOTT bacilli) were developed and evaluated in a three-phase study. In the first phase, techniques for identification of Mycobacterium species were developed by using radiometric technology and BACTEC Middlebrook 7H12 liquid medium. Based on 14CO2 evolution, characteristic growth patterns were established for 13 commonly encountered mycobacterial species. Mycobacteria belonging to the TB complex were differentiated from other mycobacteria by cellular morphology and rate of 14CO2 evolution. For further differentiation, radiometric tests for niacin production and inhibition by Q-nitro-alpha-acetyl amino-beta-hydroxy-propiophenone (NAP) were developed. In the second phase, 100 coded specimens on Lowenstein-Jensen medium were identified as members of the TB complex, MOTT bacilli, bacteria other than mycobacteria, or "no viable organisms" within 3 to 12 (average 6.4) days of receipt from the Centers for Disease Control. Isolation and identification of mycobacteria from 20 simulated sputum specimens were carried out in phase III. Out of 20 sputum specimens, 16 contained culturable mycobacteria, and all of the positives were detected by the BACTEC method in an average of 7.3 days. The positive mycobacterial cultures were isolated and identified as TB complex or MOTT bacilli in an average of 12.8 days. The radiometric NAP test was found to be highly sensitive and specific for a rapid identification of TB complex, whereas the radiometric niacin test was found to have some inherent problems. Radiometric BACTEC and conventional methodologies were in complete agreement in Phase II as well as in Phase III.

Cells, Cultured↗

The usefulness of phage typing Mycobacterium tuberculosis isolates.

Mycobacteriophage typing of Mycobacterium tuberculosis isolates was used as an epidemiologic aid in investigating the transmission of tuberculosis in community, industrial, and institutional outbreaks. The technique was also useful in other situations, e.g., documenting congenital transmission of infection and distinguishing exogenous reinfection from endogenous reactivation. Additional studies are indicated to further explore the value of phage typing for tracking the transmission of tuberculosis in the community.

Adolescent↗

Peritonitis due to a mycobacterium chelonei-like organism associated with intermittent chronic peritoneal dialysis.

Two outbreaks of peritonitis caused by a Mycobacterium chelonei-like organism--a previously unrecognized pathogen--occurred among patients receiving intermittent chronic peritoneal dialysis (CPD). In one center, five of 22 patients who had undergone CPD during a one-month period developed peritonitis caused by an M. chelonei-like organism acquired from a single contaminated automated CPD machine. In a second center, five of eight patients who had received CPD during a several-week period became infected, apparently as a result of cross-infection from contaminated machines. Seven sporadic cases of peritonitis due to an M. chelonei-like organism were also found. M. chelonei-like organisms can survive and proliferate in water and are relatively resistant to formaldehyde. Defects in the design of CPD machines and disinfection procedures were identified that may have permitted M. chelonei-like organisms to survive attempted disinfection.

Adult↗