Intracellular survival of Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease being increasingly recognized as an important cause of morbidity and mortality in many regions of the world. Several features of melioidosis suggest that B. pseudomallei is a facultative intracellular pathogen. This study was designed to assess the ability of B. pseudomallei to invade and survive in eukaryotic cells. We have shown that B. pseudomallei has the capacity to invade cultured cell lines, including HeLa, CHO, A549, and Vero cells. We have demonstrated intracellular survival of B. pseudomallei in professional phagocytic cells, including rat alveolar macrophages. B pseudomallei was localized inside vacuoles in human monocyte-like U937 cells, a histiocytic lymphoma cell line with phagocytic properties. Additionally, electron microscopic visualization of B. pseudomallei-infected HeLa cells and polymorphonuclear leukocytes confirmed the presence of intracellular bacteria within membrane-bound vacuoles. B. pseudomallei was found to be resistant to the cationic peptide protamine and to purified human defensin HNP-1.     

Initial evaluation of a continuous speech recognition program for radiology

The aims of this work were to measure the accuracy of one continuous speech recognition product and dependence on the speaker's gender and status as a native or nonnative English speaker, and evaluate the product's potential for routine use in transcribing radiology reports. IBM MedSpeak/Radiology software, version 1.1 was evaluated by 6 speakers. Two were nonnative English speakers, and 3 were men. Each speaker dictated a set of 12 reports. The reports included neurologic and body imaging examinations performed with 6 different modalities. The dictated and original report texts were compared, and error rates for overall, significant, and subtle significant errors were computed. Error rate dependence on modality, native English speaker status, and gender were evaluated by performing ttests. The overall error rate was 10.3 +/- 3.3%. No difference in accuracy between men and women was found; however, significant differences were seen for overall and significant errors when comparing native and nonnative English speakers (P = .009 and P = .008, respectively). The speech recognition software is approximately 90% accurate, and while practical implementation issues (rather than accuracy) currently limit routine use of this product throughout a radiology practice, application in niche areas such as the emergency room currently is being pursued. This methodology provides a convenient way to compare the initial accuracy of different speech recognition products, and changes in accuracy over time, in a detailed and sensitive manner.     

Effect of dexamethasone on detection of herpes simplex virus in clinical specimens by conventional cell culture and rapid 24-well plate centrifugation.

During a 4-month period, two methods for rapid detection of herpes simplex virus (HSV) were examined: (i) pretreatment of A549 cells with dexamethasone for conventional tissue culture (277 specimens) and (ii) 24-well plate centrifugation using A549 cells with and without dexamethasone pretreatment and staining with serotype-specific monoclonal antibodies (Syva Co., Palo Alto, Calif.) after incubation for 16 to 18 h (153 specimens). By conventional tube cell culture, both with and without dexamethasone, HSV was identified in 88 of 277 (32%) specimens. Significantly more specimens were positive for HSV at 24 h (46 versus 27 specimens) and at 48 h (a total of 72 versus 59 specimens) (P less than 0.0001) in dexamethasone-treated A549 cells. Of the 153 specimens tested by conventional culture and 24-well plate centrifugation, HSV was detected in 44 (29%) by conventional culture, and by 24-well plate centrifugation with and without dexamethasone, HSV was detected in 32 (21%) and 30 (20%) specimens, respectively. The sensitivity, specificity, and positive and negative predictive values of 24-well plate centrifugation with A549 cells for detection of HSV were 73 (71% without dexamethasone), 100, 100, and 90%, respectively. In conventional tube cell culture, pretreatment of A549 cells with dexamethasone results in more rapid detection of HSV. Centrifugal inoculation of dexamethasone-treated and untreated A549 cells in 24-well plates and staining with monoclonal antibodies after incubation for 16 to 18 h is an insensitive means to detect HSV in clinical specimens and should not replace conventional tube cell culture.     

Evaluation of MicroScan MIC panels for detection of oxacillin-resistant staphylococci.

Clinical isolates of staphylococci (420 Staphylococcus aureus isolates and 248 coagulase-negative staphylococci) were tested by both MicroScan MIC panels (MicroScan, West Sacramento, Calif.) and an oxacillin agar screen (Mueller-Hinton agar [Difco Laboratories, Detroit, Mich.] containing 6 micrograms of oxacillin per ml and 4% NaCl) to evaluate the ability of MicroScan to detect oxacillin-resistant strains. MicroScan panels and oxacillin agar screen plates were incubated at 35 degrees C for 24 h and at 30 degrees C for an additional 24 h. Endpoints were recorded at 24 and 48 h. By MicroScan, 23 (5.5%) and 30 (7%) S. aureus isolates and 161 (65%) and 162 (65%) coagulase-negative staphylococci were oxacillin resistant at 24 and 48 h, respectively. At both 24 and 48 h, 23 (5.5%) S. aureus isolates and 162 (65%) coagulase-negative staphylococci were resistant by the oxacillin agar screen. Five strains for which the oxacillin MIC was 2 or 4 micrograms/ml and eight strains resistant to oxacillin only at 48 h were further evaluated by broth macrodilution testing for oxacillin with and without clavulanic acid, by oxacillin and amoxicillin-clavulanic acid disk diffusion, and by oxacillin agar screen comparing Mueller-Hinton agars purchased from Difco and BBL Microbiology Systems, Cockeysville, Md. By this additional testing, all 10 S. aureus isolates and 1 of 3 coagulase-negative staphylococci examined produced increased amounts of beta-lactamase. One coagulase-negative staphylococcus appeared to be truly intermediately oxacillin susceptible. There was no significant difference in the rate of detection of oxacillin resistance between MicroScan and the agar screen. MicroScan panels should be incubated for 24 h only, because prolonged incubation caused strains producing excessive amounts of beta-lactamase to appear to be falsely oxacillin resistant.     

Use of A-549 cells in a clinical virology laboratory.

A-549 cells were compared with other cell lines for virus recovery, except from specimens submitted specifically for detection of cytomegalovirus. Of 589 specimens submitted specifically for detection of herpes simplex virus (HSV), 163 (28%) were positive for HSV--159 (97.5%) in A-549 cells and 156 (96%) in primary rabbit kidney cells. HSV cytopathic effect was identified an average of 0.6 day earlier in A-549 cells. Virus was recovered from 194 (11%) of 1,790 specimens submitted for general virus isolation. Of 40 HSV isolates, 85% were positive in A-549 cells, 72.5% were positive in MRC-5/WI-38 cells, and 42.5% were positive in HEp-2 cells. With adenovirus, 96% of 45 isolates were detected in A-549 cells, 62% were detected in HEp-2 cells, 38% were detected in MRC-5/WI-38 cells, and 31% were detected in PMK cells. Of the 76 enterovirus-positive specimens, 71% were positive in PMK cells, 62% were positive in A-549 cells, and 62% were positive in MRC-5/WI-38 cells. None of 12 respiratory syncytial virus, 14 rhinovirus, or 7 influenza A virus isolates were detected in A-549 cells. Of the cell lines examined, A-549 cells performed optimally for recovery of HSV and adenovirus, they allowed good growth of many of the enterovirus isolates, but they did not allow recovery of any of the respiratory syncytial virus, rhinovirus, or influenza A virus isolates.     

Molecular epidemiology of Legionella species by restriction endonuclease and alloenzyme analysis.

As part of an ongoing investigation into nosocomial Legionella infections at Stanford University Medical Center (SUMC), we applied the technique of restriction endonuclease analysis (REA) to determine strain differences among three species, including Legionella pneumophila, Legionella dumoffii, and Legionella micdadei. A total of 26 human and environmental water isolates from SUMC were selected for REA and compared with control strains that were not epidemiologically linked to SUMC. REA results were compared with results of alloenzyme typing, typing by monoclonal antibodies, and plasmid fingerprinting in all but L. micdadei strains. REA and alloenzyme typing showed that SUMC patient isolates were derived from distinct strains of three species. L. pneumophila strains from SUMC patients were genotypically identical to those isolated from potable water. REA was especially useful in proving that SUMC L. dumoffii patient isolates were derived from a single strain and that patients may have been exposed to a common source(s). REA typing correlated well with alloenzyme typing. These methods complement serologic typing of L. pneumophila and provide discriminating capability between strains of other Legionella species such as L. dumoffii, for which serologic types have not been identified. In addition, REA typing is somewhat easier to perform than alloenzyme typing and can be done in clinical laboratories.     

Conventional tube cell culture compared with centrifugal inoculation of MRC-5 cells and staining with monoclonal antibodies for detection of herpes simplex virus in clinical specimens.

During a 15-month period, two methods for detection of herpes simplex virus (HSV) in 699 clinical specimens were compared: (i) 24-well-plate centrifugation (24WPC) with MRC-5 cells and staining with type-specific monoclonal antibodies (Syva Co., Palo Alto, Calif.) after incubation for 16 to 18 h and (ii) conventional tube cell culture with primary rabbit kidney and A549 cells. HSV was identified by conventional tube cell culture in 165 (24%) of 699 specimens and by the 24WPC method in 116 (17%) of 699 specimens. One specimen was positive for HSV by the 24WPC method alone, compared with 50 specimens positive only by conventional cell culture (P less than 0.0001). The sensitivity, specificity, and positive and negative predictive values of the 24WPC technique with MRC-5 cells for detection of HSV in clinical specimens were 70, 99.8, 99, and 91%, respectively. Centrifugal inoculation of MRC-5 cells in 24-well plates and staining with monoclonal antibodies after incubation for 16 to 18 h is an insensitive means of detecting HSV in clinical specimens and should not replace conventional tube cell culture with primary rabbit kidney cells.     

Distributed cortical networks for focused auditory attention and distraction

We used behavioral measures and functional magnetic resonance imaging (fMRI) to study the effects of parametrically varied task-irrelevant pitch changes in attended sounds on loudness-discrimination performance and brain activity in cortical surface maps. Ten subjects discriminated tone loudness in sequences that also included infrequent task-irrelevant pitch changes. Consistent with results of previous studies, the task-irrelevant pitch changes impaired performance in the loudness discrimination task. Auditory stimulation, attention-enhanced processing of sounds and motor responding during the loudness discrimination task activated supratemporal (auditory cortex) and inferior parietal areas bilaterally and left-hemisphere (contralateral to the hand used for responding) motor areas. Large pitch changes were associated with right hemisphere supratemporal activations as well as widespread bilateral activations in the frontal lobe and along the intraparietal sulcus. Loudness discrimination and distracting pitch changes activated common areas in the right supratemporal gyrus, left medial frontal cortex, left precentral gyrus, and left inferior parietal cortex.     

Hypothalamo-adenohypophyseal-thyroid interrelationships in the developing chick embryo. VII. Immunocytochemical demonstration of thyrotrophin-releasing hormone

Thyrotrophin-releasing hormone (TRH) was demonstrated immunocytochemically in the infundibulum of the chick embryo as early as Day 4.5 of incubation. From Days 4.5 through 19.5 of embryonic development there is a gradual increase within the developing hypothalamus in the number of TRH-positive perikarya as well as the amount of immunoreactive-TRH (IR-TRH) per cell. There are no abrupt changes in either parameter during the critical time period (Days 10.5-13.5 of incubation) in the maturation of the pituitary-thyroid axis. Thus, although TRH is probably not directly responsible for the dramatic increase in the number of thyrotrophin-producing cells which occurs in the pars distalis of 10.5- to 11.5-day-old embryos (R. C. Thommes, J. B. Martens, W. E. Hopkins, J. Caliendo, M. J. Sorrentino, and J. E. Woods (1983). Gen. Comp. Endocrinol. 51, 434-443) the marked change in the activity of the pituitary-thyroid unit at this time may well reflect the response of these newly differentiated thyrothrophs to low levels of plasma TRH. This hypothesis is supported by the observations that between Days 10.5 and 11.5 the hypothalamic-adenohypophyseal-thyroid (HAT) axis is first responsive to cold (R. C. Thommes, J. B. Martens, J. B. Hopkins, D. A. Griesbach, D. J. Williams, M. J. Sorrentino, P. Wernke, and J. E. Woods. In "Proceedings, Ninth International Symposium on Comparative Endocrinology Hong Kong, 7-11 December 1981" (B. Lofts, ed.). Hong Kong Univ. Press, Hong Kong, in press) and also that the pituitary-thyroid unit exhibits a marked increase in its sensitivity to exogenous TRH (R. C. Thommes, D. J. Williams, and J. E. Woods (1984). Gen. Comp. Endocrinol. 55, 275-279).     

Differentiation between mu and kappa receptor-mediated effects in opioid drug discrimination: apparent pA2 analysis

Apparent pA2 values for the opioid antagonist, quadazocine, were used to characterize differential involvement of mu and kappa opioid receptors in the discriminative stimulus effects of opioid agonists. Rhesus monkeys were trained to discriminate s.c. injections of either codeine or ethylketazocine from sham injections. In tests of drug generalization, morphine, levorphanol and alfentanil all produced dose-dependent increases in codeine-appropriate responding, and ethylketazocine produced dose-dependent increases in ethylketazocine-appropriate responding. Quadazocine antagonized the discriminative stimulus effects of each of the agonists. Apparent pA2 values for quadazocine (and slopes of the regression lines fit to the data in "Schild Plot" analysis) were 7.8 (-1.0) with morphine, 7.7 (-1.4) with levorphanol, 7.9 (-0.92) with alfentanil and 5.7 (-0.93) with ethylketazocine. If regression line slopes were constrained to equal -1, 7.8 was the apparent pA2 value with all agonists except ethylketazocine (5.7). This difference in apparent pA2 values for quadazocine confirms that different receptors (mu and kappa, respectively) mediate the discriminative effects of opioid agonists in codeine- and ethylketazocine-trained rhesus monkeys. Also, when the antagonism data were reanalyzed separately for each individual monkey, apparent pA2 values from individual animals were found to be similar to values from other animals in the same group and to values based on grouped data.