Feasibility of a quantitative polymerase chain reaction assay for diagnosing pneumococcal pneumonia using oropharyngeal swabs.

Streptococcus pneumoniae is the most important pathogen causing community-acquired pneumonia (CAP). The current diagnostic microbial standard detects S. pneumoniae in less than 30% of CAP cases. A quantitative polymerase chain reaction (PCR) targeting autolysin (lytA) is able to increase the rate of detection. The aim of this study is validation of this quantitative PCR in vitro using different available strains and in vivo using clinical samples (oropharyngeal swabs). The PCR autolysin (lytA) was validated by testing the intra- and inter-run variability. Also, the in vitro specificity and sensitivity, including the lower limit of detection was determined. In addition, a pilot-study was performed using samples from patients (n = 28) with pneumococcal pneumonia and patients (n = 28) with a pneumonia without detection of S. pneumoniae with the current diagnostic microbial standard, but with detection of either a viral and or another bacterial pathogen to validate this test further. The intra- and inter-run variability were relatively low (SD's ranging from 0.08 to 0.96 cycle thresholds). The lower limit of detection turned out to be 1-10 DNA copies/reaction. In-vitro sensitivity and specificity of the tested specimens (8 strains carrying lytA and 6 strains negative for lytA) were both 100%. In patients with pneumococcal and non-pneumococcal pneumonia a cut-off value of 6.000 copies/mL would lead to a sensitivity of 57.1% and a specificity of 85.7%. We were able to develop a quantitative PCR targeting lytA with good in-vitro test characteristics.

Detection of carriers of the autosomal form of chronic granulomatous disease.

The NADPH:O2 oxidoreductase catalyzing the respiratory burst in activated phagocytes from healthy individuals is not operative in phagocytes from patients with chronic granulomatous disease (CGD). In a microscopic slide test using the dye nitroblue tetrazolium (NBT), carriers of X-linked CGD can be recognized by a mosaic pattern of NBT-positive and NBT-negative cells, governed by the expression of an unaffected or an affected X chromosome, respectively. Until now, it has not been possible to detect carriers of the autosomal form of CGD (other than by family studies) because all cells of these carriers stain positive in the NBT test. We have investigated whether neutrophils from carriers of autosomal CGD can be recognized by measurement of the rate of oxygen uptake upon stimulation of the cells. It was found that with the phorbol ester PMA as a stimulus, the respiratory burst is significantly lower in autosomal CGD carriers. With serum-treated zymosan as a stimulus, no difference between controls and carriers was observed. The addition of f-Met-Leu-Phe (1 microM) to PMA-activated neutrophils of control donors caused a transient increase in oxygen consumption of about 40%. Under these conditions, an increase of more than 100% was observed in neutrophils from carriers of autosomal CGD. These findings provide a simple method for the detection of carriers of the autosomal form of CGD.

Purification and partial characterization of the b-type cytochrome from human polymorphonuclear leukocytes.

Polymorphonuclear leukocytes contain an oxidase system that can be activated to produce superoxide radicals and hydrogen peroxide. A nonmitochondrial b cytochrome, functioning in the generation of these oxygen species, has been purified to apparent homogeneity from human polymorphonuclear phagocytes. After solubilization of the cytochrome with Triton X-100, the cell extract was subsequently chromatographed on Blue Sepharose and Sephacryl S-300. The final preparation was maximally purified 170-fold with a specific content of 5.33 +/- 2.03 nmol mg-1 of protein (mean +/- S.D.; n = 7) and a yield of 21 +/- 13% (n = 5). The apparent molecular mass of the nondenatured cytochrome was estimated by gel filtration to be 235 kDa. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single polypeptide was found with a molecular mass of 127 kDa. From the pyridine hemochrome spectrum 1 protoheme IX/polypeptide was calculated. The light absorbance bands of the dithionite-reduced cytochrome were found to be at 558.5 (alpha), 529 (beta), and 426 nm (Soret), and that of the oxidized cytochrome at 413.5 nm. The difference absorbance coefficients are delta epsilon (426.5 - 440 nm) = 160.6 +/- 11 mM-1 cm-1 and delta epsilon (558.5 - 542 nm) = 29.3 +/- 2 mM-1 cm-1 (mean +/- S.D.; n = 5). Carbon monoxide binds to the cytochrome in a time-dependent fashion (maximum binding after 50-60 min). The midpoint potential of the solubilized nonpurified cytochrome is identical to the cytochrome in situ (Em7.0 = -218 +/- 7 mV (mean +/- S.D.; n = 5)). However, purified cytochrome b shows a significantly decreased midpoint potential, estimated at -407 +/- 18 mV (n = 4). The protein does not contain noncovalently bound FAD or FMN, and no spectral evidence was obtained for the presence of covalently bound flavin. Preliminary amino acid analysis of the cytochrome shows a high content of hydrophilic residues.

Discrepancies in the oxygen balance of whole human neutrophils and neutrophil homogenates.

The oxygen consumption, superoxide production and hydrogen peroxide generation was studied in human neutrophils phagocytosing zymosan particles. Application of sodium azide, as an inhibitor of catalase, and/or 1,3-bis(chloroethyl)-1-nitrosourea (BCNU), as an inhibitor of glutathione reductase, led to the conclusion that neutrophils convert about half of the oxygen consumed in the respiratory burst to hydrogen peroxide; the other half is used for formation of organic peroxides, disulfide bridges, etc. These products are rapidly degraded to water by catalase and/or the glutathione redox cycle. Reduction of exogenous cytochrome C accounted for only about 15% of the consumed oxygen. Neutrophil homogenates contain a badly damaged oxidase system, because oxygen consumption and hydrogen peroxide formation were only about one-tenth of that observed with whole cells. In contrast, cytochrome-C reduction was about three times as high as that found with intact cells. Probably, cytochrome C partly reconstitutes damaged oxidase systems, thus artificially increasing the oxidase activity. We conclude that cytochrome-C reduction is not a good parameter to characterize cell-free oxidase preparations.

Effect of ascorbate on abnormal neutrophil, platelet and lymphocytic function in a patient with the Chediak-Higashi syndrome.

A diminished chemotactic response was observed with the neutrophils of a patient with the Chediak-Higashi syndrome, who was not in the accelerated phase of the disease. An abnormally low release of myeloperoxidase from these cells during phagocytosis was also noted; this resulted in a decreased iodination capacity and probably also caused the defect in the intracellular killing of bacteria by the neutrophils. The level of cyclic AMP in these cells was elevated, but decreased after treatment with ascorbate either in vitro or in vivo. During ascorbate therapy, the bactericidal activity of the neutrophils normalized, whereas the chemotactic response remained low. Nevertheless, the patient had significantly less infections during ascorbate therapy. The bleeding tendency, due to a storage-pool disorder of the Chediak-Higashi platelets, was unaffected by treatment with ascorbate. The patient's lymphocytes did not display any activity in antibody-dependent lymphocytotoxicity. This defect was not affected by treatment with ascorbate either.

Phenylglyoxal is not a selective inhibitor of phagocytosis.

In 1976, it has been reported that phenylglyoxal (C6H5COCHO) selectively inhibits endocytosis in phagocytes of rabbit and mouse. We have tested the specificity of this compound by measuring its effect on human neutrophil chemotaxis, respiration and release of lysosomal enzymes. Pretreatment of human neutrophils with 100 microgram phenylglyoxal/ml for 30 min at 37 degrees C resulted in almost complete inhibition of phagocytosis of opsonized zymosan. However, after treatment with phenylglyoxal, spontaneous mobility as well as chemotaxis of these cells towards casein, rosette formation with opsonized zymosan, stimulation of the oxidative metabolism and release of lysosomal enzymes were also severely decreased. Most of these functions were only partially restored by resuspension of the cells in a medium without phenylglyoxal. The intracellular level of ATP was not affected by phenylglyoxal, but the level of reduced glutathione was decreased. We conclude from the inhibitory action of phenylglyoxal on the stimulated oxygen consumption and its reaction with intracellular glutathione that phenylglyoxal does not necessarily act exclusively on the outside of the plasma membrane. From our studies, it follows that phenylglyoxal is not a specific inhibitor of endo- or exocytosis in human neutrophils. Phenylglyoxal can be used effectively in the bacterial-killing test of phagocytes to inhibit intracellular killing after an initial period of ingestion.

Destruction of sensitized erythrocytes by human monocytes in vitro: effects of cytochalasin B, hydrocortisone and colchicine.

The purpose of this study was to characterize the destruction of sensitized erythrocytes by human blood monocytes in vitro. The incubation in vitro of human monocytes with 51Cr-labelled human erythrocytes sensitized with IgG rhesus alloantibodies anti-D (EAIgG anti-D) resulted in release of 51Cr from the erythrocytes (lysis) as well as uptake of 51Cr-labelled erythrocytes by the monocytes (phagocytosis). The lysis of EAIgG anti-D by monocytes was not dependent on phagocytosis, because cytochalasin B, which inhibited phagocytosis of EAIgG, enhanced lysis. In contrast, hydrocortisone and colchicine inhibited lysis, but had no effect on phagocytosis. These agents did not affect binding of EAIgG anti-D to monocytes. The effect of these agents on lysis corresponded to their effect on release of lysosomal enzymes by monocytes. The release of lysosomal enzymes, when induced by EAIgG anti-D, was, likewise, enhanced by cytochalasin B and inhibited by hydrocortisone and colchicine. A significant correlation was found between lysosomal enzyme release and lysis. Together, these results strongly suggest that lysosomal enzymes, released by the monocytes when incubated with anti-D-sensitized erythrocytes, are responsible for the cytotoxic activity of these cells towards sensitized erythrocytes. The action of these enzymes only occurs over a short range, probably at the site of attachment of the erythrocyte, because only erythrocytes that were bound to the monocytes were lysed. The finding of other investigators that removal of monocytes from suspensions of human mononuclear leucocytes results in a strong reduction in the cytotoxic activity of these leucocytes towards sensitized erythrocytes in vitro. was confirmed.

Defective initiation of the metabolic stimulation in phagocytizing granulocytes: a new congenital defect.

Two patients suffering from recurrent bacterial infections were studied: a boy and a girl from one family, children of apparently healthy parents. The granulocytes of these patients were capable of normal ingestion of latex particles and DNA-anti-DNA immune complexes. When the metabolic changes in these granulocytes during phagocytosis of latex particles were studied, however, no stimulation of oxygen consumption, superoxide production, or hexose monophosphate shunt activity could be observed. Moreover, zymosan particles were not iodinated. These findings are comparable to those found in chronic granulomatous disease. In sharp contrast to the observations in this latter disease, however, a completely normal stimulation of cell metabolism was found after phagocytosis of IgG-coated latex particles or IgG aggregates. Since latex and IgG-coated latex were equally well ingested, this means that the absence of metabolic stimulation after uptake of tatexf metabolic stimulation after uptake of latex must be due to a defect in the triggering of the oxidative burst. As far as we know, this is the first time that a defect in the triggering of the metabolic stimulation during phagocytosis could be demonstrated. Moreover, these finding suggest that adherence and subsequent ingestion of particles are in themselves not sufficient for the metabolic stimulation of granulocytes.