Auramine fluorescence for acid-fast bacilli in formalin-fixed paraffin-embedded tissues.
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Benzophenoneidum↗
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Auramine fluorescence for acid-fast bacilli in formalin-fixed, paraffin-embedded tissues.
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Bacillus↗
Histologic parameters predictive of mycobacterial infection.
Tissue specimens from a wide variety of anatomic locations are frequently examined for mycobacteria using a combination of cultures and special stains. Auramine-rhodamine (AR) staining is a sensitive method for detecting acid-fast bacilli (AFB) in tissue sections. We reviewed 85 AR-positive and 275 randomly selected AR-negative biopsy specimens collected during the past 2 years at the Mayo Clinic, Rochester, Minn. Pathologic diagnoses and culture results were also reviewed. Biopsy specimens containing necrotizing granulomas yielded the highest positivity rate for AFB (61 [47.7%]), followed by nonnecrotizing granulomas (14 [17.7%]). Poorly formed granulomas (5 [16.1%]) and acute inflammation (5 [15.6%]) were less frequently positive. Cases with fibrotic or hyalinized granulomas, nonspecific chronic inflammation, nonspecific reactive or reparative changes, no significant histologic abnormality, or malignancy failed to disclose AFB. These specimens, which were consistently negative for AFB, were responsible for 25% of the samples submitted. Of the 360 tissue specimens submitted, 166 had a corresponding mycobacterial culture. Mycobacteria were cultured only from the biopsy specimens that contained necrotizing granulomas (38.2%), nonnecrotizing granulomas (32.4%), poorly formed granulomas (30.0%), or acute inflammation (15.8%). Tissues with fibrotic or hyalinized granulomas, nonspecific chronic inflammation, nonspecific reactive or reparative changes, no significant histologic abnormality, or malignancy failed to yield positive cultures. These data suggest that biopsy specimens with these latter diagnoses are inappropriate specimens for mycobacterial culture or AR staining.
Acute Disease↗
An automated optoelectronic reticulocyte counter.
Microscopic reticulocyte counting is time consuming and imprecise. A new reticulocyte counter has been developed, and the authors evaluated its utility for laboratory use. The counter, R-1000 of Sysmex-TOA Medical Electronics Company, Kobe, Japan, is based on the principles of flow cytometry. Reticulocytes are detected as fluorescent cells stained with a basic dye, auramine O, under argon-laser light. The automated count had high correlation to the manual count (r = 0.941). Linearity and reproducibility were both high. About 60 specimens were tested in one hour. Not only the reticulocyte percentage and count but also the maturity of reticulocytes was found from the intensity of the fluorescence, whether high, moderate, or slight. Normal reference values were 0.007 +/- 0.0055 (0.70 +/- 0.55%) for the reticulocytes, (4.63 +/- 1.09) X 10(9)/L for the reticulocyte count, 2.3 +/- 1.9% for highly fluorescent cells, 18.7 +/- 5.1% for moderately fluorescent cells, and 78.8 +/- 6.6% for cells with slight fluorescence. In patients with suppressed bone marrow function, such as is caused by chemotherapy, the reticulocyte fraction and count were low, and cells with slight fluorescence increased. In patients in whom bone marrow function was stimulated, such as with hemolytic anemia, the reticulocyte percentage, reticulocyte count, and highly fluorescent cells were high. Patients with chronic renal failure being treated by hemodialysis had a similar reticulocyte pattern to that in hemolytic anemia except that the reticulocyte count was decreased. Results for elderly patients were not different from those of healthy young controls. Some patients with a normal reticulocyte count and percentage had numerous highly fluorescent cells, perhaps because of hemolytic anemia not yet identified. Automated reticulocyte counting provides reliable data, so such measurement should be useful for analysis of the kinetics of red blood cells and for the study of the pathogenesis of anemia.
Adult↗
Factors affecting the clinical value of microscopy for acid-fast bacilli.
In order to assess the clinical value of microscopy for acid-fast bacilli (AFB), the results of 3,207 clinical specimens submitted for mycobacterial smear and culture were analyzed. Mycobacteria grew from 176 (5.5%) of the specimens, 95 (54%) of which were Mycobacterium tuberculosis. Although the overall sensitivity of the smear was low (33%), 65% of respiratory specimens yielding M. tuberculosis had positive AFB smears. Furthermore, 96% of patients with pulmonary tuberculosis from whom more than one specimen was processed had at least a single positive AFB smear. Smear sensitivity correlated well with quantitative growth; 89% of specimens yielding greater than or equal to 50 colonies per slant were smear positive. Specificity of the AFB smear was high; 89% of smear-positive specimens had positive cultures. After the results from culture-negative patients known to have active tuberculosis were eliminated from the analysis, the specificity of a positive smear rose to 98.3%. When the results of all specimens from each patient were considered in toto, the AFB smear had a predictive value of greater than or equal to 96%.
Aniline Compounds↗
Morphological study of bacteria of the respiratory system using fluorescence microscopy of Papanicolaou-stained smears with special regard to the identification of Mycobacteria sp.
In Papanicolaou-stained smears certain structures such as nucleoli, Pneumocystis carinii, Charcot-Leyden crystals, bacteria and fungi show a brilliant fluorescence. The morphological characteristics of microorganisms which can be detected by this system, especially mycobacteria, are described. This screening method offers the possibility of providing the clinician with a provisional diagnosis within hours. Proof of the nature of the organisms should be obtained by culture.
Adult↗
Liquid substitution: a versatile procedure for SEM specimen preparation of biological materials without drying or coating.
Certain liquids with a very low vapour pressure, such as glycerol or triethylene glycol, can be used to infiltrate biological specimens so that they may be observed in the scanning electron microscope (SEM) without drying. The conductive properties of the fluids allow specimens to be examined either uncoated or with very thin coatings. The advantages of liquid substitution include the retention of lipids, waxes, loose particles, and surface contaminants. Since the procedure does not require expensive equipment, it offers an alternative to critical point drying or cryopreparation. For certain types of specimens, liquid substitution may represent the best preparation procedure. In addition, the fluids themselves may be imaged directly in the SEM, or indirectly by cathodoluminescence following labelling with fluorochromes.
Animals↗
Interactions of heteroaromatic compounds with nucleic acids. A - T-specific non-intercalating DNA ligands.
In the present paper we report the results of a study on the base specificity and affinity of eight dyes potentially able to interact with DNA. These compounds include four triphenylmethane dyes used in histochemistry, auramine, "Hoechst 33258" and two acridines substituted with t-butyl groups. They were selected with regard to their inability to intercalate between the base pairs of helical polynucleotides due to structural limitations. Hydrodynamic studies performed with the DNA complexes of crystal violet and Hoechst 33258 confirmed our assumptions that compounds of this type bind to the outside of DNA. The main results from DNA binding studies indicate that the triphenylmethane dyes except p-fuchsin are bound with high preference to two adjacent A - T pairs while Hoechst 33258 seems to need three A - T pairs as the binding site. Model studies with synthetic polynucleotides revealed that not only a sequence of A - T pairs, but also their structural arrangement in a helix, is crucial for the high affinities observed for most of the ligands when interacting with natural DNA. Methyl green and Hoechst 33258 can be used for increasing the resolution power of cesium chloride density gradients for DNAs with different (A + T) content.
Acridines↗
Auramine O as a conformational probe to study glyceraldehyde 3-phosphate dehydrogenase.
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Aniline Compounds↗
Binding of ligands to horse liver alcohol dehydrogenase lacking zinc ions at the active sites.
The zinc-deficient enzyme binds the fluorescence probes for the enzyme substrate pocket (auramine O, 13-ethylberberine, chlorprothixene and acridine orange) more tightly than the native enzyme, whereas 1-anilinonaphthalene 8-sulphonic acid is bound with comparable affinity. The use of fluorescence probes as reporter ligands revealed that the formation of binary complexes between the zinc-deficient enzyme and aldehydes is possible (as with the native enzyme) and confirmed an increased affinity of coenzymes to the modified enzyme. The absence of catalytic zinc ions brings about a loss of the essential stabilization effect in simultaneous NADH and aldehyde binding to liver alcohol dehydrogenase. 2,2'-Bipyridine, which chelates the active-site zinc ion in the native enzyme, is bound rather loosely to the same site as aldehydes, auramine O and ethylberberine in the case of the zinc-depleted enzyme. The stopped-flow measurements showed that the pH dependence of auramine O and ethylberberine binding to native and zinc-depleted enzyme is essentially similar. These data are compatible with the presence of ionizable groups in the surroundings of the bound probes. This group might be either His-67, bound to the zinc ion, or the zinc-liganding water molecule in the case of the native enzyme (pK close to 9), or the free His-67 residue in the case of the zinc-deficient enzyme (pK about 8).
2,2'-Dipyridyl↗
Dissociation of outer-sphere water is rate-limiting for the binding of ligands in the active site of horse liver alcohol dehydrogenase.
The association of imidazole and auramine O to native horse-liver alcohol dehydrogenase [Zn(II)LADH] and active-site specifically cobalt(II)-substituted horse-liver alcohol dehydrogenase [Co(II)LADH], respectively, has been investigated. In all cases [except imidazole binding to Zn(II)LADH in the presence of auramine O] the association rates approached an upper limit (kmax). The kmax values were compared for the metal ligands imidazole (monodentate), 1,10-phenanthroline and 2,2'-bipyridine (bidentate; see also the preceding paper), and for auramine O which does not coordinate to the catalytic metal ion. Independent of the large differences in their structure and metal-bonding capability, all these compounds exhibit common, maximum, limiting rate constants of about 60 s-1 and 200 s-1 for Co(II)LADH and Zn(II)LADH, respectively. These results demonstrate that kmax is strongly dependent on the catalytic metal ion but not on the ligand. The absence of spectral changes in the d-d transitions of the catalytic Co(II) ion upon auramine O binding to Co(II)LADH indicates that the rate-limiting step is not accompanied by a major conformational change. Finally, it is concluded that reactions in the inner coordination sphere of the catalytic metal ion (i.e. the metal-bound water molecule) are not responsible for the step characterized by kmax. We propose the rate-limiting step to consist of the dissociation of one or several water molecules from the second coordination sphere of the catalytic metal ion in the active site of LADH in its open conformation.
2,2'-Dipyridyl↗
Replication and long-term persistence of bovine and human strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga.
Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohn's disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists.
Acanthamoeba↗
Immunofluorescence detection of Cryptosporidium oocysts in fecal smears.
An indirect fluorescent antibody (IFA) procedure was developed for the detection of Cryptosporidium sp. oocysts in human, nonhuman primate, and bovine fecal smears. The procedure, which takes about 90 min to perform, involves the use of a rabbit antiserum against Cryptosporidium oocysts isolated from dairy cattle. Cross-specificity testing of the IFA method revealed no reactivity with yeasts, various amoebae, Giardia lamblia, Chilomastix sp., or Blastocystis sp. and only very weak cross-reactivity with coccidian oocysts of other genera. IFA detection of oocysts in human and nonhuman primate fecal smears was far more sensitive than was dimethyl sulfoxide-carbolfuchsin staining. Moreover, IFA detection was comparable in sensitivity to auramine O staining with samples of high oocyst concentration and somewhat more sensitive than auramine O with samples containing relatively few oocysts. The IFA procedure may be useful in the clinical diagnosis of human and animal cryptosporidiosis and also in the detection of oocysts in environmental samples.
Animals↗
Comparison of three methods for detection of Cryptosporidium oocysts in a low-prevalence population.
Four hundred fecal specimens which had been received for routine ova and parasite examination were concentrated by Formalin-ether sedimentation. Sediments were examined as saline and iodine-stained wet preparations and were stained with rhodamine-auramine O and a commercially available monoclonal fluorescent-antibody stain for oocysts of Cryptosporidium species. Examination with the fluorescent stains detected cryptosporidia in both positive specimens (0.5% prevalence), and routine direct wet-preparation examination detected cryptosporidia in one of them. Detection of only low numbers of positive specimens in our nonrisk population argues against routine use of specific and expensive stain reagents.
Animals↗
Detection of acid-fast bacilli in concentrated primary specimen smears stained with rhodamine-auramine at room temperature and at 37 degrees C.
Many laboratory workers prefer the rhodamine-auramine method of staining acid-fast bacilli (AFB) in primary specimen smears rather than carbol fuchsin stains because the stain is more readily interpreted and yields greater sensitivity. The increasing incidence of AFB infections serves as an impetus to optimize the rhodamine-auramine stain. A total of 782 primary smears were evaluated blindly by the rhodamine-auramine method at both room temperature and 37 degrees C. Thirty-five smears (4.5%) were positive for AFB, 30 were positive by both methods, and 5 were positive at 37 degrees C only. Room temperature staining detected only 85.7% of the positive primary smears. Of the 30 smears positive by both methods, 13 (43.3%) had equal numbers of AFB on both smears, 13 (43.3%) had more AFB on the smear stained at 37 degrees C, and 4 (13.3%) had greater numbers of AFB on the smear stained at room temperature. No smears were positive only when stained at room temperature. The increasing diagnostic emphasis placed on the primary smear underscores the importance of optimizing AFB smear methods, and rhodamine-auramine staining at 37 degrees C enhances the detection of AFB compared with conventional staining at room temperature.
Benzophenoneidum↗
Use of UV ParaLens adapter for detection of acid-fast organisms.
Auramine-stained mycobacterial smears from 136 clinical specimens were interpreted by using the UV ParaLens adapter (Beckton Dickinson), and results were compared with smear interpretations using a traditional fluorescent microscope and culture. The sensitivity and specificity of the ParaLens were 84 and 93%, respectively. Smears yielding discrepant results were overstained by the Kinyoun method. Overall, the sensitivity of auramine-stained smears interpreted with the UV ParaLens was comparable to that of Kinyoun-stained smears.
Benzophenoneidum↗
Phenolic acridine orange fluorescent stain for mycobacteria.
A new fluorescence acid-fast staining method with acridine orange as the specific stain is presented. Only two reagents are required: the acridine orange-specific stain and a destaining-counterstaining reagent. Compared with auramine fluorescence acid-fast staining, there was less nonspecific staining of non-acid-fast debris which fluoresced a pale green contrasting color to provide a background in which to search for the red-to-orange fluorescing acid-fast bacilli. The results of the study indicate that the acridine orange method is superior to the auramine method in detecting acid-fast bacilli in specimen smears.
Acids↗
Direct detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis in auramine-rhodamine-positive sputum specimens by real-time PCR.
Our objective was to evaluate the feasibility of a molecular assay based on a real-time PCR technique, carried out with a LightCycler instrument (Roche Biochemicals), to identify Mycobacterium tuberculosis bacilli and to detect rifampin and isoniazid resistance in DNA extracts from sputum samples. We studied three genes: rpoB, which is associated with rifampin resistance, and katG and inhA, which are associated with isoniazid resistance. A total of 205 sputum samples collected from 108 patients diagnosed with pulmonary tuberculosis with positive auramine-rhodamine-staining (AR) sputum samples, were tested. The sensitivities of the LightCycler PCR assay for the positive AR specimens was 97.5% (200 of 205) for rpoB and inhA genes and 96.5% (198 of 205) for the katG gene. For the total number of patients tested, the sensitivity was 100% (108 of 108 patients) for rifampin, whereas the sensitivity was 98.1% (106 of 108 patients) for isoniazid. Full agreement was found with the Bactec MGIT 960 method and the genotype inferred from the LightCycler data for rifampin. The phenotypic method for isoniazid reported 13 resistant strains (> or = 0.1 microg/ml). In seven (53.8%) strains there was a concordance between both methods, but we found that six (46.2%) strains reported as resistant by the phenotypic method were determined to be susceptible by real-time PCR. For the 75 strains reported as susceptible by the phenotypic method, the concordance with the LightCycler data was 100%. Our results demonstrate that rifampin-resistant M. tuberculosis could be detected in DNA extracted from auramine-rhodamine-positive sputum samples in a single-tube assay that took less than 3 h to perform for a collection of auramine-rhodamine-positive specimens obtained from patients with culture-documented pulmonary tuberculosis. Similarly, this occurs in half of the isoniazid-resistant M. tuberculosis DNA extracted from auramine-rhodamine-positive specimens.
Antitubercular Agents↗