Performance of indirect immunofluorescence assay, immunochromatography assay and reverse transcription-polymerase chain reaction for detecting human respiratory syncytial virus in nasopharyngeal aspirate samples.

Comparison of the use of indirect immunofluorescence assay (IFA), immunochromatography assay (ICA-BD) and reverse transcription-polymerase chain reaction (RT-PCR) for detecting human respiratory syncytial virus (HRSV) in 306 nasopharyngeal aspirates samples (NPA) was performed in order to assess their analytical performance. By comparing the results obtained using ICA-BD with those using IFA, we found relative indices of 85.0% for sensitivity and 91.2% for specificity, and the positive (PPV) and negative (NPV) predictive values were 85.0% and 91.2%, respectively. The relative indices for sensitivity and specificity as well as the PPV and NPV for RT-PCR were 98.0%, 89.0%, 84.0% and 99.0%, respectively, when compared to the results of IFA. In addition, comparison of the results of ICA-BD and those of RT-PCR yielded relative indices of 79.5% for sensitivity and 95.4% for specificity, as well as PPV and NPV of 92.9% and 86.0%, respectively. Although RT-PCR has shown the best performance, the substantial agreement between the ICA-BD and IFA results suggests that ICA-BD, also in addition to being a rapid and facile assay, could be suitable as an alternative diagnostic screening for HRSV infection in children.

Functional alterations of human neutrophils by medium-chain triglyceride emulsions: evaluation of phagocytosis, bacterial killing, and oxidative activity.

Medium-chain triglyceride (MCT) and long-chain triglyceride (LCT) emulsions currently used in nutritional therapy were evaluated for their in vitro effect on neutrophil oxidative metabolism, phagocytosis, and bacterial killing activities. Neutrophils from healthy adult male volunteers were assessed after blood incubation with commercially available fat emulsions containing LCT, MCT, or a mixture of 50% MCT and 50% LCT at a final triglyceride concentration of 20 mg/ml. It was observed that MCT-containing emulsions stimulated nitroblue tetrazolium (NBT) dye reduction by neutrophils as determined by a cytochemical NBT test performed directly on whole blood. This effect was dose dependent. However, after lipid removal by cell washing, the MCT-treated neutrophils showed decreased production of hydrogen peroxide (H2O2) and NBT reduction in response to bacterial lipopolysaccharide or phorbol myristate acetate stimuli as well as impaired phagocytosis and killing of Staphylococcus aureus. In contrast, the LCT emulsion did not alter any of the neutrophil functions evaluated. The present data suggest that MCTs elicit the oxidative metabolism of neutrophils, probably by phagocytosis of fat particles and, depending on the lipid concentration, this effect may not be reversible, leading to impairment of the cellular response to subsequent membrane stimuli.

Influence of medium-chain triglyceride-based lipid emulsion on rat polymorphonuclear cell functions.

Intravenous lipid emulsions are used as energy and essential fatty acids sources. There are controversial reports postulating in vitro and in vivo inhibitory effects of long-chain triglycerides (LCT) upon the blood polymorphonuclear leukocytes (PMNL) functions. In the present study the in vivo and in vitro effects of LCT and a physical mixture of medium- and long-chain triglycerides (MCT/LCT) emulsions were investigated on select PMNL functions, i.e., chemotaxis, phagocytosis, and bacterial killing. Blood from 20 rats was incubated with LCT, MCT, MCT/LCT, and saline, respectively. MCT-containing emulsions exhibited an inhibitory effect on all PMNL functions investigated, whereas LCT exerted an effect on the phagocytic index only. The administration of a parenteral supply of LCT, MCT/LCT, and saline for 30 h followed by saline infusion for 14 h in discontinuous mode did not influence any of the investigated PMNL functions. Similarly, continuous infusion over 44 h at increasing infusion rates up to 1.5 mL/h did not affect the PMNL functions. The obvious difference between in vitro and in vivo response of the PMNL model emphasizes the necessity for continuous monitoring of in vivo conditions. Appropriate interpretation of the data requires continuous circumspection and consideration of trials in a clinical setting.

Effect of total parenteral nutrition with different lipid emulsions of human monocyte and neutrophil functions.

Parenteral nutrition (TPN) with lipid emulsions is claimed to be associated with impaired monocyte (M) and neutrophil (N) functions. Long-chain triglycerides (LCT) and a mixture containing 50% medium-chain triglycerides (MCT) and 50% LCT, currently used in nutritional therapy with TPN, were evaluated for their ex vivo effects on human N and M chemotaxis, phagocytosis, bacterial killing, and oxidative metabolism by nitroblue tetrazolium reduction test. Cell functions were examined in a randomized, crossover, blind trial in 10 malnourished patients with gastric cancer. Prior to the operation (2 wk), central TPN (40 kcal/kg) with 25% of caloric energy provided as LCT or MCT/LCT emulsion was infused over 48 h. After the crossover period fat-free TPN was given over 48 h. Function tests were done for N and M before and after each lipid emulsion infusion. Every cell function test performed for each patient was controlled by another test done in healthy adult volunteers and the results were compared with the normal range of values previously established for a healthy adult population. All the patients completed the studies without complications. Crossover validity was statistically established. Bacterial killing was the only function reduced in neutrophils after LCT emulsion (% killed bacteria = 79.0 +/- 8.5 versus 67.4 +/- 19.2; P < 0.05), although this function remained within the normal range values in 80% of the patients. In conclusion, the lipid emulsions did not affect any monocyte functions and only moderately decreased neutrophil bacterial killing.

Application of a fluorochrome-lysostaphin assay to the detection of phagocytic and bactericidal disturbances in human neutrophils and monocytes.

The evaluation of phagocytic and microbicidal activities of the blood neutrophils has been recognized as one of the important tools for investigating phagocytic dysfunctions in patients with recurrent infections. In the present study, these activities were examined in neutrophils and monocytes from healthy adults and patients affected by primary phagocytic dysfunctions by using a modified fluorochromic microbicidal assay, discriminating simultaneously the extracellular adherence, ingestion and intracellular killing of Staphylococcus aureus Cowan I. The assay employs acridine orange staining, as described in Bellinati-Pires et al. (1989) (AO assay), but was modified by the addition of an alternative leukocyte treatment with 0.5 U/ml of lysostaphin (LS) for 5 min at 37 degrees C, after phagocytosis (AO-LS assay). The LS treatment was standardized to eliminate staphylococci adhered to the outer surface of the phagocytes without affecting the determination of intracellular live or dead bacteria, as demonstrated in normal neutrophils and monocytes. Our purpose in this study was to compare AO and AO-LS assays in order to evaluate the effect of LS on the determination of actually ingested staphylococci and to provide a means for improving the fluorochromic assay for detecting phagocytic defects, as well as bactericidal disturbances. By using the AO-LS assay, decreased ingestion of staphylococci by neutrophils in Chediak-Higashi Syndrome (CHS) was demonstrated. However, increased staphylococci adherence, as well as ingestion, was observed in neutrophils or monocytes from chronic granulomatous disease (CGD) patients, comparing AO and AO-LS assays. Bactericidal defect, which is a common feature in CHS and CGD, was detected in neutrophils or monocytes in both assays. We emphasize that such alterations were deduced by comparing the patients' results with those obtained from their respective normal controls and with the normal range of values previously established for 160 healthy adults. No alteration was observed in hyper IgE syndrome phagocytes. Despite the possible penetration of LS into the leukocytes, as stated in other studies, we concluded that a short period of phagocyte incubation with this enzyme increased the sensitivity of the fluorochromic assay to detect phagocytic defect without affecting the determination of the bactericidal activity. Moreover, comparations between AO and AO-LS assays may be important in the study of the initial pathways of staphylococci phagocyte interaction, including adherence by non-phagocytic receptors.

Effect of medium- and long-chain triglycerides on human neutrophil migration.

There is some controversy concerning the effect of intravenous long-chain triglyceride (LCT) emulsions on the phagocytic system and little is known about the effect of medium-chain triglyceride (MCT)-containing emulsions. We evaluated the chemotaxis and random migration of human neutrophils from 18 healthy adults after preincubation with the following fat emulsions: LCT, MCT and a mixture of 50% MCT and 50% LCT (MCT/LCT). Leukocyte-rich plasma (4 x 10(6) cells/ml) was diluted 4:1 (v/v) with commercial fat emulsions (LCT, MCT, or MCT/LCT, 1:1) or saline and tumbled at 20 cycles/min for 30 min at 37 degrees C. The final composition of the emulsion was 20 mg/ml fat, 0.24% egg yolk lecithin, and 0.5% glycerol and the dispersion was made isotonic by adding NaCl. In a second set of experiments, the LCT and MCT concentrations were adjusted to be equimolar. Leukocyte viability was > or = 95% after exposure to the treatment with fat emulsions. For emulsions with the same weight of each fat, random migration and chemotaxis of neutrophils were unaffected by the LCT emulsion but there was a significant decrease in both chemotaxis and random migration in MCT-(79 and 74%) or MCT/LCT-treated (60 and 56%) neutrophils. Similar results were obtained when LCT and MCT were equimolar. These results demonstrate an inhibitory effect of MCT on two human neutrophil functions which may be dose dependent.

Phagocytosis and killing of epidemic methicillin-resistant Staphylococcus aureus by human neutrophils and monocytes.

Staphylococcus aureus is a pathogen that has been associated with nosocomial infections since the preantibiotic era. Since the introduction of antibiotics in medical practice in the 1940 s, methicillin-resistant S. aureus (MRSA) strains have been emerging in various parts of the world. In view of the important role of the phagocytic system in the defense against this bacteria, we decided to study phagocytosis by neutrophils and monocytes of an epidemic MRSA strain in São Paulo, Brazil, in comparison with methicillin-sensitive strains. Complement system opsonins are fundamental for efficient ingestion of the resistant and sensitive strains by both types of phagocytes. We found no association of the opsonic requirement of the MRSA strain with the multiresistance phenotype. On the other hand, the MRSA strain was found to be more resistant to the effector mechanisms of neutrophils than both sensitive strains when opsonized with fresh serum, despite the phagocytosis results. This fact suggests that the intracellular killing of S. aureus is an additional parameter of bacterial virulence, but new approaches must be implemented to study the interactions of this MRSA strain with phagocytes in order to investigate the possible factors involved in its behavior in response to neutrophil effector mechanisms.

Delayed phagocytosis and bacterial killing in Chediak-Higashi syndrome neutrophils detected by a fluorochrome assay. Ultrastructural aspects.

The few studies already published about phagocyte functions in Chediak-Higashi syndrome (CHS) has stated that neutrophils present slow rate of bacterial killing but normally ingest microorganisms. In the present study, both phagocytosis and killing of Staphylococcus aureus were verified to be delayed in neutrophils from two patients with CHS when these functions were simultaneously evaluated by a fluorochrome phagocytosis assay. Electron microscopic examination showed morphologic differences among neutrophils from CHS patients and normal neutrophils regarding the cytoplasmic structures and the aspects of the phagolysosomes. It was noteworthy the presence of giant phagolysosomes enclosing bacteria in active proliferation commonly observed in CHS neutrophils after 45 min of phagocytosis, which corresponded with the impaired bactericidal activity of these leukocytes. The present results suggest that phagocytosis may also be defective in CHS, and point out to the sensitivity of the fluorochrome phagocytosis assay and its application in clinical laboratories.

[The effect of lipid emulsions on the mechanisms of organic defense in infectious injury]. / Efeito de emulsões lipídicas sobre mecanismos de defesa orgânica na agressão infecciosa.

For treatment of metabolic derangements in infective states intravenous lipidic emulsions have been used. Their use is however not harmless existing reports on fat inhibiting the function of blood polymorphonuclear leukocytes and macrophages. The purpose of the research reported herewith was to study in rats the effect of new intravenous lipidic emulsions containing medium chain triglycerides and long chain triglycerides and compare it with the effect of long chain triglycerides emulsion on function of polymorphonuclear leukocytes (chemotaxis, phagocytosis and bactericidal activity). The intraperitoneal implant of an E. coli capsule was used for the study. The transfusions of both lipidic emulsions in septic rats have not altered functions of polymorphonuclear leukocytes when compared with saline infusion. However there was found hepatic steatosis, hypertrophy and presence of fat globules in the Kupffer cells in rats infused with medium chain and long chain triglyceride emulsions. Sequential blood cultures obtained from rats infused with the emulsions showed increased bacterial growth with medium chain triglyceride emulsion. There was no significant difference between the rats that received both lipid infusions and those that received saline infusions as to the mortality. Our experimental study suggests that the use of fat emulsions in infective states be done with care and monitoring of seric triglycerides and steroids.

Identification of highly transformable wheat genotypes for mass production of fertile transgenic plants.

The efficiency of wheat biolistic transformation systems strongly depends on the bombardment parameters, the condition of the donor plant, and the plant genotype chosen for the transformation process. This paper analyzes the transformation efficiency of the 129 wheat sister lines generically called 'Bobwhite', originally obtained from the cross 'Aurora'//'Kalyan'/'Bluebird 3'/'Woodpecker'. A number of factors influencing the transformation were examined, such as the ability to produce embryogenic callus, regeneration in selection medium, and overall transformation performance. Of the 129 genotypes evaluated, eight demonstrated transformation efficiencies above 60% (60 independent transgenic events per 100 immature embryos bombarded). Among the eight genotypes identified, we studied agronomic characteristics such as earliness to identify the most adaptable line(s) for different lab conditions. 'Bobwhite' SH 98 26 was identified as a super-transformable wheat line.

Performance of indirect immunofluorescence assay, immunochromatography assay and reverse transcription-polymerase chain reaction for detecting human respiratory syncytial virus in nasopharyngeal aspirate samples

Comparison of the use of indirect immunofluorescence assay (IFA), immunochromatography assay (ICA-BD) and reverse transcription-polymerase chain reaction (RT-PCR) for detecting human respiratory syncytial virus (HRSV) in 306 nasopharyngeal aspirates samples (NPA) was performed in order to assess their analytical performance. By comparing the results obtained using ICA-BD with those using IFA, we found relative indices of 85.0 percent for sensitivity and 91.2 percent for specificity, and the positive (PPV) and negative (NPV) predictive values were 85.0 percent and 91.2 percent, respectively. The relative indices for sensitivity and specificity as well as the PPV and NPV for RT-PCR were 98.0 percent, 89.0 percent, 84.0 percent and 99.0 percent, respectively, when compared to the results of IFA. In addition, comparison of the results of ICA-BD and those of RT-PCR yielded relative indices of 79.5 percent for sensitivity and 95.4 percent for specificity, as well as PPV and NPV of 92.9 percent and 86.0 percent, respectively. Although RT-PCR has shown the best performance, the substantial agreement between the ICA-BD and IFA results suggests that ICA-BD, also in addition to being a rapid and facile assay, could be suitable as an alternative diagnostic screening for HRSV infection in children.

Phagocytosis and killing of epidemic methicillin-resistant Staphylococcus aureus by human neutrophils and monocytes

Staphylococcus aureus is a pathogen that has been associated with nosocomial infections since the preantibiotic era. Since the introduction of antibiotics in medical practice in the 1940 s, methicillin-resistant S. aureus (MRSA) strains have been emerging in various parts of the world. In view of the important role of the phagocytic system in the defense against this bacteria, we decided to study phagocytosis by neutrophils and monocytes of an epidemic MRSA strain in São Paulo, Brazil, in comparison with methicillin-sensitive strains. Complement system opsonins are fundamental for efficient ingestion of the resistant and sensitive strains by both types of phagocytes. We found no association of the opsonic requirement of the MRSA strain with the multiresistance phenotype. On the other hand, the MRSA strain was found to be more resistant to the effector mechanisms of neutrophils than both sensitive strains when opsonized with fresh serum, despite the phagocytosis results. This fact suggests that the intracellular killing of S. aureus is an additional parameter of bacterial virulence, but new approaches must be implemented to study the interactions of this MRSA strain with phagocytes in order to investigate the possible factors involved in its behavior in response to neutrophil effector mechanisms.

Effect of medium and long chain triglycerides on human neutrophil migration

There is some controversy concerning the effect of intravenous long-chain triglyceride (LCT) emulsions on the phagocytic system and little is known about the effect of medium-chain triglyceride (MCT) containing emulsions. We evaluated the chemotaxis and random migration of human neutrophils from 18 healthy adult after preincubation with the following fat emulsions: LCT, MCT and a mixture of 50% MCT and 50% LCT (MCT/LCT). Leukocyte-rich plasma (4 x 10 6 cells/ml) was diluted 4:1 (v/v) with commercial fat emulsions (LTC, MCT, or MCT?LCT, 1:1) or saline and tumbled at 20 cycles?min for 30 min at 37 grade C. The final composition or the emulsion was 20 mg/ml fat, 0.24% egg yolk lecithin, and 0.5% glycerol and the dispersion was made isotonic by adding NaCl. In a second set of experiments, the LCT and MCT concentrations were adjusted to be equimolar. Leukocyte viability was * 95% after exposure to the treatment with fat emulsions. For emulsions with the same weight of each fat, random migration and chemotaxis of neutrophils were unaffected by the LCT emulsion but there was a significant decrease in both chemotaxis and random migration in MCT- (79 and 74%) or MCT/LCT-treated (60 and 56%) neutrophils. Similar results were obtained when LCT and MCT were equimolar. These results demonstrate an inhibitory effect of MCT on two human neutrophil functions which may be dose dependent