Phagocytosis and killing of Mycobacterium avium complex by human neutrophils. (1/36)

Organisms belonging to the Mycobacterium avium complex (MAC) cause life-threatening bacteremia in immunocompromised patients. Monocytes and macrophages are thought to be responsible for ingestion and killing of MAC. However, it has been suggested that neutrophils may play a role in the early immune response to MAC infection. Here, neutrophils in autologous plasma were incubated (at 0 and 37 degrees C) with M. avium labeled with Auramine O, a potent fluorochrome. Neutrophil phagocytosis was measured by flow cytometry. Neutrophils incubated at 37 degrees C showed an increase in fluorescence over time with a maximum at 15 min, whereas neutrophils on ice showed no time-dependent increase in FL1. At 15 min Fl 1 at 37 degrees C was twice as high as FL1 at 0 degrees C. Examination under the fluorescent microscope showed multiple intracellular fluorescent mycobacteria. Results in nine independent experiments showed time-dependent decrease of colony-forming units in neutrophil-associated live M. avium. Significant killing was observed within 30 min and was complete by 120 min. Observation by electron microscopy clearly confirmed the presence of intraphagosomal MAC, both intact and with evidence of degradation. These data demonstrate that MAC is rapidly phagocytized and killed by human neutrophils. The newly established flow cytometry method should be useful in further studies of neutrophil function and of the role of G-CSF and other cytokines in MAC disease.  (+info)

Lessons from a proficiency testing event for acid-fast microscopy. (2/36)

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.  (+info)

Interactions of heteroaromatic compounds with nucleic acids. A - T-specific non-intercalating DNA ligands. (3/36)

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.  (+info)

Optimisation of acid fast smears for the direct detection of mycobacteria in clinical samples. (4/36)

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.  (+info)

Direct detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis in auramine-rhodamine-positive sputum specimens by real-time PCR. (5/36)

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.  (+info)

Comparison of the COBAS AMPLICOR MTB and BDProbeTec ET assays for detection of Mycobacterium tuberculosis in respiratory specimens. (6/36)

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.  (+info)

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. (7/36)

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.  (+info)

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. (8/36)

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.  (+info)