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PubMed2005

Comparison of the sodium hydroxide specimen processing method with the C18-carboxypropylbetaine specimen processing method using independent specimens with auramine smear, the MB/BacT liquid culture system, and the COBAS AMPLICOR MTB test.

A study was performed to diagnose tuberculosis by smear, culture, and nucleic acid amplification. The study was comprised of two independent arms. Each arm used a different specimen processing method; in one arm, all specimens were processed with N-acetyl-l-cysteine-sodium hydroxide, and in the other arm, all specimens were processed with C(18)-carboxypropylbetaine and lytic enzymes. In each arm, all processed sediments were split for analysis by auramine smear, by culture using the MB/BacT liquid culture system and solid media, and by nucleic acid amplification using the COBAS AMPLICOR MTB test. In the N-acetyl-l-cysteine-sodium hydroxide arm, 1,468 specimens were analyzed: 65 were smear positive; 88 and 42 were culture positive for Mycobacterium tuberculosis and nontuberculous mycobacteria, respectively; and 103 were PCR positive. Relative to cultures positive for M. tuberculosis, the sensitivity and specificity of the smear were 68.2% and 99.6%, respectively, and those of PCR were 75.0% and 97.3%, respectively. In the C(18)-carboxypropylbetaine study arm, 1,423 specimens were analyzed: 44 were smear positive; 82 and 31 were culture positive for M. tuberculosis and nontuberculous mycobacteria, respectively; and 91 were PCR positive. The sensitivity and specificity of the smear were 48.8% and 99.7%, respectively, and those of PCR were 78.0% and 98.0%, respectively. When the two arms were compared, C(18)-carboxypropylbetaine specimen processing significantly increased the number of smear-negative and culture-positive specimens and significantly increased the PCR sensitivity among this same group of specimens while at the same time significantly reducing the inhibition rate.

Anti-Infective Agents↗
PubMed2005

Comparison of the COBAS AMPLICOR MTB and BDProbeTec ET assays for detection of Mycobacterium tuberculosis in respiratory specimens.

The performances of the BDProbeTec ET (Becton Dickinson) and COBAS AMPLICOR MTB (Roche) were retrospectively evaluated for detecting Mycobacterium tuberculosis complex in various respiratory specimens. The BACTEC and MGIT liquid culture system (Becton Dickinson) was used as a reference method. A total of 824 respiratory specimens, comprised of sputa, bronchoalveolar lavage fluid, and bronchial and tracheal aspirates from 580 patients, were evaluated. Out of 824 clinical specimens, 109 specimens from 43 patients were culture positive for M. tuberculosis. Of these 109 specimens, 67 were smear positive, 85 were positive by the COBAS AMPLICOR MTB test, and 94 were positive by the BDProbeTec ET. Of the 715 culture-negative specimens, 17 were positive by the auramine staining, 11 were positive by the COBAS AMPLICOR MTB test, and 12 were positive by the BDProbeTec ET. After discrepancy analysis and review of the patients' clinical data, 130 specimens from 50 patients were considered "true-positive" specimens. This resulted in the following sensitivities: microscopy, 61.5%; COBAS AMPLICOR MTB test, 78.0%; and BDProbeTec ET, 86.2%. The specificities of each system, based on the clinical diagnosis, were 99.7% for microscopy, 99.9% for the COBAS AMPLICOR MTB test, and 99.9% for the BDProbeTec ET. The data presented represent a considerable number of specimens evaluated with a considerable number of culture- and auramine-positive and culture-positive and auramine-negative results and therefore give a realistic view of how the data should be interpreted in a daily routine situation. Specifically, the data with regard to the culture-positive and auramine-negative specimens are useful, because in a routine situation, auramine-negative specimens are sometimes accepted, on clinical indications, to be analyzed by an amplification method.

Benzophenoneidum↗
PubMed2005

Improved sensitivity of sputum smear microscopy after processing specimens with C18-carboxypropylbetaine to detect acid-fast bacilli: a study of United States-bound immigrants from Vietnam.

The goal of this study was to evaluate the effect of the specimen-processing method that uses the detergent C18-carboxypropylbetaine (CB-18) on the sensitivity of acid-fast bacillus (AFB) staining. Vietnamese immigrants with abnormal chest radiographs provided up to three sputum specimens, which were examined for acid-fast bacilli by use of direct auramine and Ziehl-Neelsen staining. The remaining sputum was split; half was cultured, and the other half was incubated with CB-18 for 24 h, centrifuged, and examined for AFB by both staining methods. CB-18 processing improved the sensitivity of AFB staining by 20 to 30% (only differences in auramine sensitivity were statistically significant) but reduced specificity by approximately 20% (P < 0.05). These findings have direct utility for overseas migrant tuberculosis screening programs, for which maximizing test sensitivity is a major objective.

Adult↗
PubMed1976

Comparison of machine and manual staining of direct smears for acid-fast bacilli by fluorescence microscopy.

Comparisons were made in Lusaka and in London between manual staining and staining in an automatic machine with auramine-phenol of direct smears of sputum and other types of specimen for acid-fast bacilli. No evidence was obtained of carry-over of acid-fast bacilli from positive to negative smears during machine staining. There was improved contrast between bacilli and the background in smears prepared with the machine.

Benzophenoneidum↗
PubMed1994

Evaluation of a safe sputum processing method for detecting tuberculosis.

AIMS: To evaluate a safe sputum processing method for detection of tuberculosis in developing countries. METHODS: A sample processing method was developed in which acid fast bacilli were killed with 1% sodium hypochlorite and concentrated by flotation on a layer of xylene before staining by the Ziehl Neelsen or auramine O methods. RESULTS: Best results were obtained by auramine O staining after flotation. Staining by the Ziehl Neelsen method after flotation gave better results than direct Ziehl Neelsen staining without flotation. CONCLUSIONS: The flotation method with Ziehl Neelsen staining offers advantages for smear preparation in the tuberculosis control programmes of developing countries.

Bacteriological Techniques↗
PubMed2003

Optimisation of acid fast smears for the direct detection of mycobacteria in clinical samples.

AIMS: Despite its long history, the acid fast smear remains unstandardised. Technical variations in both the preparation of clinical material and subsequent staining mean that smear sensitivity relative to culture may vary from 50% to over 80%. This study assessed the sensitivity of acid fast microscopy at each of five stages of sample preparation and by both commonly used staining methods. METHODS: Sputum samples thought for varying reasons to be highly likely to be culture positive were used to prepare a series of smears in which the effects of digestion (liquefaction), concentration (centrifugation), and decontamination (sodium hydroxide) could be assessed, together with a comparison of staining by the auramine/phenol and Ziehl-Neelsen techniques. RESULTS: The most effective method for the demonstration of acid fast organisms in sputum was found to be an auramine phenol stain applied to a liquefied, concentrated sample and examined before the decontamination process. CONCLUSIONS: The auramine phenol stain applied to a liquefied, concentrated sample and examined before the decontamination process is the most effective method for the demonstration of acid fast organisms in sputum.

Bacteriological Techniques↗
PubMed1976

Low-affinity interaction of ethanol with the fluorescent complex between horse liver alcohol dehydrogenase and auramine O.

Quenching of the fluorescence of the complex between horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase (EC 1.1.1.1) and auramine O complex is inconsistent with a simple competitive displacement of auramine O by ethanol. Instead, the action of ethanol requires an explanation in terms of a solvent effect, or the formation of an enzyme-auramine O-ethanol ternary complex. The latter complex would have to be the low-affinity variety similar to the enzyme-NADH-ethanol ternary complex encountered in the kinetic system.

Alcohol Oxidoreductases↗
PubMed1976

The reliability of gastric smears by auramine-rhodamine staining technique for the diagnosis of tuberculosis.

From 1972 to 1974, all sputum specimens and gastric aspirate specimens submitted to the University of Michigan Laboratory for acid-fast smear and culture were studied. Specimens were paired for culture and smear results using the auramine-rhodamine staining technique. Of 1,893 patients, 75 patients without prior antituberculous therapy were found to have either a positive smear or a positive culture of either sputum or gastric material. The data analyzed by patient source revealed the following. (1) Staining sputum with auramine-rhodamine is a clinically reliable technique for detecting pulmonary tuberculosis. It demonstrates a sensitivity of 78 per cent and a relative fraction of false positive smears of only 11 per cent. (2) Staining gastric-aspirated material by the auramine-rhodamine technique is not a clinically reliable method as a routine procedure for the detection of pulmonary tuberculosis, because of a sensitivity of only 58.8 per cent and a relative fraction of false-positive smears of 33 per cent. (3) In the absence of sputum in suspected clinical granulomatous disease, quantified gastric smears may be helpful. In this study, when more than 6 organisms per high power field were found, the patient's sputum or gastric material yielded a pathogenic mycobacterium on culture.

Aniline Compounds↗
PubMed1979

Re-evaluation of sputum staining for the diagnosis of pulmonary tuberculosis.

Data were collected at 2 teaching institutions, University Hospital, University of Michigan (UMH) and University of California at Los Angeles (UCLA) Hospital, to evaluate the usefulness of the auramine-rhodamine fluorochrome stain in the diagnosis of pulmonary tuberculosis in hospitalized patients. The patients studied had received no prior therapy, and their sputum specimens were positive for a pathogenic Mycobacterium by microscopy or culture or both. The true-positive smear rate was 0.88 at UMH and 0.83 at UCLA; the relative false-positive smear rate was 0.12 at UMH and 0.17 at UCLA. The sensitivities at UMH and UCLA were 0.78 and 0.51, respectively, and the specificity was greater than 0.99 at both. The data suggest that sputum stained by the fluorochrome method is useful and reliable for the diagnosis of pulmonary tuberculosis, because the false-positive rates are acceptably low in hospitalized patients.

Benzophenoneidum↗
PubMed1980

A case of Mycobacterium avium infection: early detection by the auramine-O fluorescent technique.

Infection due to Mycobacterium avium is rare in man and its definitive diagnosis is based upon culture, usually taken from a cervical lesion. The lack of systemic involvement, the negative history for contact with tuberculosis, the normal chest roentgenogram and the fact that Mycobacterium avium complex grows slowly on standard Mycobacterial media, make it difficult to diagnosis. The purpose of this communication is to re-emphasize the possibility of infection by group III Mycobacterium complex in the differential diagnosis of children with cervical lymphadenitis and to describe the important use of Auramine-O fluorescent stain in the early detection of this disease.

Aniline Compounds↗
PubMed2000

Analysis of a simplified concentration sputum smear technique for pulmonary tuberculosis diagnosis in rural hospitals.

Rural hospitals in Africa face the challenge of an increasing number of pulmonary tuberculosis (PTB) cases, much of it related with the AIDS pandemic. The main diagnostic tool in these hospitals is the direct sputum smears. Sensitivity of direct sputum smears is low, especially in cases of AIDS related PTB. Several concentration methods have been described but none is routinely used in Zimbabwe. Fluorescence microscopy of auramine stained samples increases sensitivity and saves laboratory time but it is not an appropriate technique for rural African hospitals. In a rural hospital of southwest Zimbabwe, we studied the sensitivity and appropriateness of a simplified concentration method. The sensitivity against culture was significantly higher with the concentration method (80%) than with the classical direct smear method (57%). However, when sputum is not adequate (with high number of squamous epithelial cells, meaning upper airway origin), the sensitivity of this method is low. A combination of both methods is proposed to increase the sensitivity of PTB diagnosis in rural hospitals.

Adult↗
PubMed2006

A testing scheme for the detection of Mycobacterium avium subsp. paratuberculosis in bovine feces utilizing the ESP para-JEM liquid culture system.

A testing scheme for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in broth cultures of bovine fecal samples carried out in ESP para-JEM System was evaluated. The scheme included acid-fast staining (on signal-positive and signal-negative samples), and confirmation by PCR for 2 MAP-specific targets and subculture of all acid-fast positive PCR-negative samples. Two hundred and fifty bovine fecal samples were evaluated for the presence of MAP using this scheme. Thirty-seven (15%) of 250 fecal samples had a positive culture result when the proposed testing scheme was used, compared to 14 (6%) positive results when using the standard ESP para-JEM protocol (requiring samples to have a positive signal from the system, a positive acid-fast stain, and a positive IS900 PCR result), and 20 (8%) positives when conventional culture was performed on Herrold egg yolk (HEY) media. A preliminary comparison of real-time and conventional PCR on DNA extracted from 15 MAP-positive broth cultures by 3 different protocols suggested that conventional PCR may be a better choice for the confirmation of the presence of MAP in the liquid cultures than real-time PCR.

Animals↗
PubMed2001

Lessons from a proficiency testing event for acid-fast microscopy.

OBJECTIVES: To evaluate the routine performance and the technical parameters of different acid-fast staining methods: Kinyoun, Ziehl-Neelsen (ZN), auramine, and auramine-rhodamine. DESIGN AND PARTICIPANTS: The performance of 167 laboratories was analyzed using prestained and unstained slides. SETTING: Laboratories holding New York State permits. RESULTS: The results revealed that Kinyoun's cold carbol fuchsin method is inferior to both the ZN and fluorochrome (auramine and/or auramine-rhodamine) methods. Even though 91% of the participants used commercial staining kits, the study identified unexpected errors concerning the concentration of carbol fuchsin, time for staining and counterstaining, and the concentration of acid alcohol for decolorization, which may significantly influence the sensitivity. Besides these findings, the present study showed that the examination of < 300 view fields may also decrease the sensitivity of acid-fast microscopy. In addition, we found that the sensitivity and specificity of the ZN and fluorochrome methods are comparable if the procedural standards are followed. CONCLUSIONS: The strict and ongoing quality control of the "simple to perform" acid-fast microscopy and the immediate review of commercially available staining kits are necessary. Because of the rapidity of the fluorochrome method, laboratories with large specimen numbers should use this technique. In all other cases, the ZN method should be used. Moreover, all clinicians should be aware of the method of acid-fast microscopy used and the proficiency of the laboratory in performing the assay.

Benzophenoneidum↗
PubMed1998

Reticulocytes and reticulated platelets: simultaneous measurement in whole blood by flow cytometry.

Reticulated platelets are a fraction of newly released circulating elements characterized by a residual amount of RNA. It has been suggested that the reticulated platelet count, providing an estimate of thrombopoiesis in the same way as erythrocyte reticulocyte count is a measure of erythropoiesis, may be useful in the study of thrombocytopenic disorders. Reticulated red cells and platelets can be analyzed by flow cytometry using specific stains for nucleic acids such as Thiazole Orange and Auramine-O. The aim of our work was to perform the simultaneous evaluation of reticulated elements in whole blood using a standard flow cytometer and to correlate the results obtained with a dedicated cytometer. A group of 14 patients with abnormal absolute reticulocyte counts (range 1.1-11%) and a group of 41 patients showing a platelet discrimination error when analyzed with a dedicated flow cytometer (Sysmex R1000) were enrolled. Linear amplification of both scatter and fluorescence was used to perform reticulocyte count. A gate was set on platelet dimensions, and logarithmic amplification of scatter and fluorescence was used to count reticulated platelets. A good correlation was obtained both for results of reticulocyte count (r2 = 0.9825) and for reticulated platelets (r2 = 0.8717) between our method and those using dedicated instruments. These data show that reticulated platelet count may be easily introduced in clinical laboratories that routinely perform reticulocyte count by flow cytometry.

Benzophenoneidum↗
PubMed1980

Combined haematoxylin, eosin and fluorescent stain for paraffin sections.

Combined staining is achieved by adding Auramine O to 2 of the steps of the standard H & E staining procedure. Auramine O confers fluorescence to mucin, reticulin, elastin and protein aggregates without altering the H & E colouring pattern. The technique is designed as a substitute for some special stains.

Aniline Compounds↗