Comparison of BACTEC MYCO/F LYTIC and WAMPOLE ISOLATOR 10 (lysis-centrifugation) systems for detection of bacteremia, mycobacteremia, and fungemia in a developing country

In less-developed countries, studies of bloodstream infections (BSI) have been hindered because of the difficulty and costs of culturing blood for bacteria, mycobacteria, and fungi. During two study periods (study period I [1997] and study period II [1998]), we cultured blood from patients in Malawi by using the BACTEC MYCO/F LYTIC (MFL), ISOLATOR 10 (Isolator), Septi-Chek AFB (SC-AFB), and Septi-Chek bacterial (SC-B) systems. During study period I, blood was inoculated at 5 ml into an MFL bottle, 10 ml into an Isolator tube for lysis and centrifugation, and 10 ml into an SC-B bottle. Next, 0.5-ml aliquots of Isolator concentrate were inoculated into an SC-AFB bottle and onto Middlebrook 7H11 agar slants, chocolate agar slants, and Inhibitory Mold Agar (IMA) slants. During study period II, the SC-B and chocolate agar cultures were discontinued. MFL growth was detected by fluorescence caused by shining UV light (lambda = 365 nm) onto the indicator on the bottom of the bottle. During study period I, 251 blood cultures yielded 44 bacterial isolates. For bacteremia, the MFL was similar to the Isolator concentrate on chocolate agar (34 of 44 versus 27 of 44; P, not significant [NS]), but more sensitive than the SC-B bottle (34 of 44 versus 24 of 44; P = 0.05). For both study periods combined, 486 blood cultures yielded 37 mycobacterial and 13 fungal isolates. For mycobacteremia, the sensitivities of the MFL and Isolator concentrate in the SC-AFB bottle were similar (30 of 37 versus 29 of 37; P, NS); the MFL bottle was more sensitive than the concentrate on Middlebrook agar (30 of 37 versus 15 of 37; P = 0.002). For fungemia, the MFL bottle was as sensitive as the SC-B bottle or Isolator concentrate on chocolate agar or IMA slants. We conclude that the MFL bottle, inoculated with just 5 ml of blood and examined under UV light, provides a sensitive and uncomplicated method for comprehensive detection of BSI in less-developed countries.     

Controlled comparison of BacT/ALERT FAN aerobic medium and BATEC fungal blood culture medium for detection of fungemia

Yeasts are an increasingly common cause of nosocomial bloodstream infections. Methods for their detection are many; controlled comparisons are few. The vented FAN aerobic blood culture medium has been shown to be superior to the standard BacT/ALERT aerobic medium for the detection of fungemia as well as bacteremia. The BACTEC selective fungal medium (FM) (BD Biosciences, Sparks, Md.) allowed detection of more episodes of fungemia than did a resin-containing medium with equal volumes of blood cultured. Therefore, we compared vented FAN to FM for the ability to recover fungi from the blood of patients who were at increased risk of having fungemia. From 5,109 cultures processed for which both FAN and FM bottles were adequately filled, fungi were recovered from 87 cultures. Of these, 47 were detected with both bottles, 12 were detected with FAN only, and 28 were detected with FM only (P < 0.05). FAN was the first bottle positive for 36 of the 47 cultures for which both bottles yielded the same fungus, whereas the FM bottle was the first bottle positive for 11 cultures (P < 0.001). A total of 54 episodes of fungemia were identified, with 40 detected by both media, 4 detected only by FAN, and 10 detected only by FM (P value, not significant). We conclude that the vented FAN aerobic bottle is comparable to the FM bottle for detection of episodes of yeast infection but has the added benefit of detecting bacteria.     

Morphometry of right ventricular hypertrophy induced by myocardial infarction in the rat.

The growth response of the right ventricle was studied in rats following ligation of the left coronary artery, which produced infarcts comprising approximately 40% of the left ventricle. A month after surgery the weight of the right ventricle was increased 30%, and this hypertrophic change was characterized by a 17% wall thickening, consistent with the 13% greater diameter of myocytes. Myocardial hypertrophy was accompanied by an inadequate growth of the microvasculature that supports tissue oxygenation. This was seen by relative decreases in capillary luminal volume density (-27%) and capillary luminal surface density (-21%) and by an increase in the average maximum distance from the capillary wall to the mitochondria of myocytes (19%). In contrast, measurements of the mean myocyte volume per nucleus showed a proportional enlargement of these cells (32%), from 16,300 cu mu in control animals to 21,500 cu mu in experimental rats. Quantitative analysis of the right coronary artery revealed a 33% increase in its luminal area, commensurate with the magnitude of ventricular hypertrophy.     

Lymphatic endothelial cell identity is reversible and its maintenance requires Prox1 activity

The activity of the homeobox gene Prox1 is necessary and sufficient for venous blood endothelial cells (BECs) to acquire a lymphatic endothelial cell (LEC) fate. We determined that the differentiated LEC phenotype is a plastic, reprogrammable condition that depends on constant Prox1 activity for its maintenance. We show that conditional down-regulation of Prox1 during embryonic, postnatal, or adult stages is sufficient to reprogram LECs into BECs. Consequently, the identity of the mutant lymphatic vessels is also partially reprogrammed as they acquire some features typical of the blood vasculature. siRNA-mediated down-regulation of Prox1 in LECs in culture demonstrates that reprogramming of LECs into BECs is a Prox1-dependent, cell-autonomous process. We propose that Prox1 acts as a binary switch that suppresses BEC identity and promotes and maintains LEC identity; switching off Prox1 activity is sufficient to initiate a reprogramming cascade leading to the dedifferentiation of LECs into BECs. Therefore, LECs are one of the few differentiated cell types that require constant expression of a certain gene to maintain their phenotypic identity.     

Neutrophils Injure Bronchial Epithelium after Ozone Exposure

Neutrophil (PMN) influx is an early, prominent finding in the airways of humans after experimental inhalation of ozone (O3), however the potential for PMN to contribute to epithelial injury in this setting is unknown. Bronchial epithelial cells of the human BEAS 2B R1.4 cell line or primary human bronchial epithelial cells underwent DNA labeling by incubation with BrdU. Monolayers were exposed to O3 (0.05 to 1 ppm) or filtered air for 60 min., and subsequently incubated with PMN for 2 h. Epithelial cell cytolysis was significant only in BEAS exposed to O3 and co-cultured with PMN. Apoptosis was maximal in BEAS exposed to O3 + PMN. Primary bronchial epithelial cells were resistant to injury; no cytolysis was detected, and apoptosis was detected only after treatment with 10 mM H₂O₂. Neutrophils may increase damage to the respiratory epithelium after O3 exposure, but primary bronchial epithelial cells are resistant to PMN and ozone induced injury.     

Absence of aluminium in neurofibrillary tangles in Alzheimer''s disease

Using the new technique of nuclear microscopy, aluminium is not detected in pyramidal neurons in brain tissue from Alzheimer's disease (AD) patients. The analytical technique of nuclear microscopy can simultaneously image and analyse features in unstained and untreated tissue sections. In tissue which had been previously subjected to conventional procedures such as fixation and osmication, aluminium was observed in both neurons and surrounding tissue. This result shows that the analysis of tissue prepared using conventional chemical techniques may produce contamination or elemental redistribution, and supports our previous investigations which implied that aluminium is not involved in the aetiology of AD. In addition, significant increases in iron, phosphorus and sulphur concentrations were noted between neurons from Alzheimer tissue and neurons from age-matched controls, and between the supporting Alzheimer tissue and supporting control tissue, implying an overall increase in these elements. No significant increase in calcium was observed between neurons from Alzheimer tissue and neurons from age-matched controls.     

Hydrogen peroxide induces DNA single strand breaks in respiratory epithelial cells

The respiratory epithelium is often exposed to oxidant gases, including ozone from photochemical smog and toxic oxygen metabolites released from neutrophils recruited in conditions of airway inflammation. We evaluated DNA single strand break formation by alkaline elution as a measure of oxidant-induced DNA damage to bronchial epithelial cells. Human AdenoSV-40-transformed bronchial epithelial cells (BEAS), subclone R1.4 or nonhuman primate bronchial epithelial cells were cultured in growth factor supplemented Ham's F12 medium on polycarbonate filters. DNA was labeled by incubation with [³H]thymidine. Cells were incubated for 1 h in HBSS or HBSS and increasing concentrations of hydrogen peroxide (H₂O₂). Cells incubated in H₂O₂ demonstrated dose-dependent increases in strand break formation, and BEAS cells were more sensitive to H₂O₂-induced injury than primary bronchial epithelial cells. The addition of catalase or preincubation of cells with the iron chelator desferoxamine prevented H₂O₂-induced strand breakage. DNA strand break formation may be an important mechanism of oxidant injury in respiratory epithelial cells.This work was supported by NIEHS grant ES-00628 and California Primate Research Center Base grant. Portions of this work were presented at the American Thoracic Society annual meeting, May 1992, Miami, Florida.     

Methicillin-resistant Staphylococcus aureus: laboratory detection methods in use in the Republic of Ireland and Northern Ireland

There is no universally agreed laboratory protocol for the detection of methicillin-resistant Staphylococcus aureus (MRSA) and hence a variety of approaches are used. As part of an all-island survey of MRSA in the Republic of Ireland (the South) and Northern Ireland (the North), a questionnaire was circulated to 14 participating laboratories in the North and 49 in the South, to determine the methods used to isolate MRSA from clinical specimens, identify S. aureus and test for susceptibility to methicillin. Almost two-thirds (64%) of laboratories in the North but only 16% of laboratories in the South use enrichment culture. There is heavy reliance on commercial kits to confirm the identification of S. aureus in the South but all laboratories in the North use the staphylocoagulase test. More than 90% of all laboratories use a disc method for susceptibility testing and 71% of laboratories in the North supplement this with the E-test; however, a range of methicillin disk concentrations are in use. There is a need to review current laboratory methods used to detect MRSA, with follow-up audit on their implementation. Additional resources may be needed in some laboratories to comply with revised guidelines, and reference facilities are required to assess new commercially available techniques and to confirm the identification of unusual or difficult strains.     

Nuclear expression of p53, p21 and cyclin D1 is increased in bronchioloalveolar carcinoma

AIMS: The objectives of this study were: (1) to determine, using immunohistochemistry, the level of expression of the cell cycle factors p53, p21 and cyclin D1 in a group of bronchioloalveolar carcinomas (BACs), and to compare these data to relevant published data for lung carcinoma; (2) to determine if higher expression rates for these factors in BAC were associated statistically with advanced clinical stage, greater tumour size, tobacco abuse, and/or BAC subtype; (3) to seek, using Fisher's exact t-test and paired data groups, any significant associations within the expression data for p53, p21 and cyclin D1. METHODS AND RESULTS: A panel of monoclonal antibodies against p53, p21 and cyclin D1 was applied to 19 bronchioloalveolar carcinomas (17 surgical pathology cases and two autopsies) from the tissue archives of St. Louis University. These immunohistochemical stains were graded on a semiquantitative scale according to the prevalence of nuclear staining within the tumour (< 10% positive cells = 0, 10-25% = 1+, 25-50% = 2+, 50-75% = 3+ and 75-100% = 4+). Six of 19 (32%) of BACs showed 1+ or greater p53 positivity, six of 19 (32%) showed 1+ or greater nuclear cyclin D1 positivity, and nine of 19 (47%) of BACs showed 1+ or greater p21 nuclear positivity. A statistically significant correlation was found between p53 and cyclin D1 expression (P = 0.046, Fisher's exact t-test), but not between p53 and p21, or between p21 and cyclin D1. No statistically significant association was found between the cell cycle factor expression data and subtype of BAC (mucinous vs. nonmucinous), tumour diameter, clinical stage or tobacco-use history. CONCLUSIONS: BACs show p53 immunostain positivity at a frequency similar to that published for p53 mutations in lung adenocarcinomas in general. Cyclin D1 and p21 nuclear expression characterizes a significant proportion of BACs, with cyclin D1 and p53 expression showing a statistically significant association. Aberrations in p53, p21, and cyclin D1 expression may be important in the development of a significant proportion of BACs.