Urine cultures in a long-term care facility (LTCF): time for improvement.

BACKGROUND: Urinary tract infections (UTIs) are the most prevalent infections in long-term care facilities (LTCFs). Numerous studies have described the problem of inadequate UTI diagnosis and treatment. We assessed the role of urine cultures in the diagnosis and treatment of UTIs in a LTCF. METHODS: In a 370-bed non-academic LTCF a retrospective assessment of antibiotic (AB) prescriptions for UTIs and urine cultures was performed from July 2014 to January 2016. The reasons why physicians, including 11 nursing home physicians and 2 junior doctors, ordered urine cultures were recorded using questionnaires. RESULTS: During the study period, 378 residents were prescribed 1672 AB courses; 803 were for UTIs. One hundred and fifty-five urine cultures were obtained from 135 residents; 66 of these cultures were performed on the same day as ABs were prescribed (8% of all prescriptions for UTI), while 89 were not. There was a discrepancy between the actions that seemed logical based on the culture results and the actions that were actually taken in 75% of the cases. In these cases, initial AB treatment was not adjusted when the isolated microorganism was resistant to the AB prescribed, the urine culture was positive and no ABs had previously been administered, or ABs were prescribed and no microorganism was isolated. The most frequent reason for ordering a urine culture was to confirm the diagnosis of a UTI. CONCLUSION: In the majority of patients, AB therapy was not adjusted when the urine culture results suggested it may be appropriate. The physicians were erroneously convinced that UTIs could be diagnosed by a positive urine culture.

Characteristics of a pathogenic molecular clone of an end-stage serum-derived variant of simian immunodeficiency virus (SIV(F359)).

End-stage simian immunodeficiency virus (SIV) isolates are suggested to be the most fit of the evolved virulent variants that precipitate the progression to AIDS. To determine if there were common characteristics of end-stage variants which emerge from accelerated cases of AIDS, a molecular clone was derived directly from serum following in vivo selection of a highly virulent SIV isolate obtained by serial end-stage passage in rhesus monkeys (Macaca mulatta). This dominant variant caused a marked cytopathic effect and replicated to very high levels in activated but not resting peripheral blood lymphocytes. Furthermore, although this clone infected but did not replicate to detectable levels in rhesus monocyte-derived macrophages, these cells were able to transmit infection to autologous T cells upon contact. Interestingly, although at low doses this end-stage variant did not use any of the known coreceptors except CCR5, it was able to infect and replicate in human peripheral blood mononuclear cells homozygous for the Delta 32 deletion of CCR5, suggesting the use of a novel coreceptor. It represents the first pathogenic molecular clone of SIV derived from viral RNA in serum and provides evidence that not only the genetic but also the biological characteristics acquired by highly fit late-stage disease variants may be distinct in different hosts.

A strategy for cloning infectious molecular clones of retroviruses from serum or plasma.

To enable biological characterisation of lentiviral variants which emerge during infection and development of AIDS, a method was developed to construct molecular clones from circulating simian immunodeficiency virus (SIV) particles present in as little as 20 microl of serum from infected rhesus monkeys. This technique uses a long distance RT-PCR method optimised for the amplification of partly overlapping 5-kb SIV (half genome) amplimers. Ligation of the genome halves resulted in the construction of full-length clones which, after transfection, were able to replicate well in rhesus peripheral blood mononuclear cells (PBMCs) and in various human T-cell lines inducing syncytia. In addition to the study of molecular cloned virus quasispecies emerging in circulation as a result of immune escape, this method may also be applied to obtain entire genes or full-length molecular clones. These clones may be present in other extracellular body fluids such as urine, saliva, tears, lymph, and bronchial or cerebral spinal fluid. Genes amplified in this way can be inserted quickly in new recombinant expression vectors and may then be applied for DNA vaccination approaches.

Direct amplification and cloning of up to 5-kb lentivirus genomes from serum.

To produce large cDNA strands from biological samples containing limited numbers of template molecules, it may be necessary to minimize both nonspecific primer attachment in first-strand synthesis and secondary structure in RNA molecules. Failure to do so could result in the accumulation of shortened cDNA strands and therefore may reduce the yield of large cDNA molecules, sometimes below detection level. We show that 5.0-kb cDNA fragments can be generated from simian immunodeficiency virus RNA in a specific reverse transcription (RT)-PCR by increasing the stringency of the primer-annealing conditions, followed by the elimination of excess free primer. Since this method utilizes a relatively long primer in the first-strand cDNA synthesis, it is possible to heat-denature the nonspecific RNA/primer complexes and RNA secondary structure without dissociating the primer from the specific template. In contrast to classic RT assays, in which an excess of primer is annealed to denatured RNA just prior to and during reverse transcription at relative low temperatures (37 degrees-42 degrees C), this method eliminates false priming. To optimize the yield and fidelity of full-length cDNA molecules, two PCR amplifications are first performed using both Taq and Pfu polymerase, followed by Pfu alone in the second amplification.

Characterization of the natural immune response of rhesus monkey CD4+ve T cells to the bacterial antigen streptolysin O (SLO).

Rhesus monkeys show a high proliferative T cell response to the bacterial exotoxin SLO without prior immunization. The present study was undertaken to characterize this naturally present SLO-responsiveness with particular emphasis on CD4+ve reactive T cells. It is demonstrated that the frequency of SLO-reactive cells in the circulation.ranges between 1 in 75 and 1 in 610 CD4+ve T cells as determined with limiting dilution analysis. It is also shown that induction of a good proliferative response requires Mhc-DR matching between T cell and the antigen presenting cells (APC). Stable and DR-restricted SLO-specific CD4+ve T cell lines were generated from CD8 depleted peripheral blood mononuclear cells (PBMC). The SLO-reactive CD4+ve cell lines are tentatively characterized as Th1-like based on the predominant production of interferon-gamma (IFN-gamma) over IL-4, although this seems contradicted by the IL-4 dependent growth of the lines.

Human interleukin-3 contains a discontinuous zinc binding domain.

The primary structure of human interleukin-3 contains two amino acid consensus sequences at Glutamate 22- Histidine 26 and Histidine 95-Histidine 98, that are characteristic for zinc binding proteins. Therefore, the hypothesis was tested that human interleukin-3 binds zinc specifically by either one or both sequences. Protein dotblotting, followed by probing with radioactive zinc demonstrated specific zinc binding of interleukin-3. Metal specificity was confirmed by competition experiments with 12 other divalent- and trivalent metal ions. Protease treatment combined with plasma desorption mass spectrometry was used to localize the zinc binding domain. Specific zinc binding was restricted to a fragment composed of Threonine 11-Lysine 28 and Asparagine 80-Lysine 100. It was found to decrease by a factor of five when either of these two amino acid stretches was missing. It is concluded that human interleukin-3 is a zinc binding protein. Interleukin-3 zinc binding capacity is largely determined by both moieties of the protein that contain the consensus sequences. In addition we propose that the zinc binding of hIL-3 is involved in (de)phosphorylation of the hIL-3 receptor.

Cellular origins of human polymeric and monomeric IgA: enumeration of single cells secreting polymeric IgA1 and IgA2 in peripheral blood, bone marrow, spleen, gingiva and synovial tissue.

Using modified ELISA and spot-ELISA, which permit the parallel determination of heavy chain subclass and the presence of covalently linked J chain, we analysed IgA found in cell culture supernatants or secreted by individual cells from peripheral blood, spleen, bone marrow, gingiva and synovial tissue, with respect to its polymeric or monomeric IgA form (pIgA, mIgA) and IgA1 or IgA2 subclass. The ELISA for determination of J chain in tissue culture supernatants was specific and highly sensitive (detection limit in pg). The results demonstrated that IgA1-producing cells predominated in the tissues examined, and that J chain could be detected in association with the majority of IgA1 and IgA2 secreted by individual cells. With respect to the frequency of cells secreting polymeric, J chain-containing IgA, only 20-30% of cells from the bone marrow were engaged in the synthesis of PIgA. In other tissues the frequency of cells secreting pIgA1 and pIgA2 was considerably higher. Peripheral blood mononuclear cells secreting pIgA2 were easily inducible during stimulation with T cell-dependent pokeweed mitogen, whereas Epstein-Barr virus-transformed cells secreted preferentially mIgA1. When the frequencies of pIgA-, pIgA1- or pIgA2-secreting cells (determined by spot-ELISA technique) from different tissues were correlated with the proportion of pIgA to mIgA (and IgA subclasses) secreted in tissue culture supernatants, data obtained suggest that many individual IgA-producing cells could be engaged in simultaneous secretion of mIgA and pIgA.

Antibodies to a short synthetic peptide related to the hinge segment of human IgG3 recognizes thermally or fixative induced conformational changes in the human IgG3 molecule.

A synthetic decapeptide (SP) was used to produce a murine monoclonal antibody specific for the human IgG3 molecule. Recognition of the IgG3 determinant is heat- and fixation-sensitive in ELISA and immunoenzyme cytology, respectively. The antibody specifically recognizes a sequence from the hinge region of IgG3, but only when subtle alterations in the conformation are induced by mild heating (greater than 40 degrees) and subsequent stabilization by means of electrostatic interactions in solid-phase assays or by fixation with formalin acetic acid mercury chloride. The structure of the human IgG3 molecule is especially sensitive to microenvironmental influences, as can be concluded from its behaviour under various physicochemical conditions. To this, we add that the immunogenic determinants in the structure of relatively flexible parts of this protein can be severely altered by the routine application of fixation methods. It is shown that these changes can also be of importance in the recognition of antigen by monoclonal antibodies.

IgA antibodies against phenolic glycolipid I from Mycobacterium leprae in serum of leprosy patients and contacts: subclass distribution and relation to disease activity.

The anti-PGL-I IgA response against phenolic glycolipid I (PGL-I) a specific surface antigen of Mycobacterium leprae, was demonstrated to be essentially of the IgA1 subclass in sera from leprosy patients and contacts. Anti-PGL-I IgA1 mean levels were found to increase significantly from the tuberculoid toward the lepromatous pole of the leprosy disease spectrum, thus resembling the predominating anti-PGL-I IgM response. Furthermore, anti-PGL-I IgA1 values were shown to increase significantly with increasing bacillary load, measured as bacillary index (BI) from skin biopsies. However, a number of BI negative leprosy patients recorded elevated anti-PGL-I IgA1 levels possibly reflecting a persistence of disease activity. Three of 28 household or family contacts of leprosy patients were detected seropositive for anti-PGL-I IgA1. Thus, our results suggest that anti-PGL-I IgA1 may be considered as an additional parameter for the early detection of infection with M. leprae.

Elevated production of polymeric and monomeric IgA1 by the bone marrow in IgA nephropathy.

In bone marrow cultures of 15 patients with primary IgA nephropathy we found significantly (P = 0.02) increased synthesis of both monomeric and polymeric IgA1 compared to 23 controls, by using high performance liquid chromatography (HPLC) fractionation of culture supernatants. The relative contribution of polymeric to total IgA1 produced was not different for the two groups. Two-color immunofluorescence studies of the percentage of bone marrow IgA1 plasma cells able to bind secretory component in vitro showed no difference between patients and controls. In the sera of patients with primary IgA nephropathy the relative contribution of IgA1 polymers to total IgA1 was also similar to controls. These results indicate that in IgA nephropathy, the increased IgA production in the bone marrow is restricted to the IgA1 subclass. The production of both monomeric and polymeric IgA1 is increased in patients during a quiescent phase of the disease.

The bone marrow as production site of the IgA deposited in the kidneys of patients with IgA nephropathy.

Patients with primary IgA nephropathy have increased plasma levels of polymeric IgA1 and deposits of IgA1 in their kidneys. The origin of this material is unknown. The production of IgA and its subclasses was investigated in the bone marrow of 14 patients and 19 controls using two colour immunofluorescence and tissue culture. Patients had an increase in the percentage of plasma cells containing IgA (45.8 +/- 7.2 mean +/- s.d.) compared to controls (40.1 +/- 10.5) (P = 0.08). IgA plasma cells containing subclass IgA1 were significantly (P less than 0.01) increased in patients (89.9 +/- 2.7%) compared to controls (84.1 +/- 6.7%). Correspondingly IgA plasma cells containing subclass IgA2 were significantly decreased (P less than 0.01) in patients (7.4 +/- 3.0%) compared to controls (13.5 +/- 5.9%). Production of IgA in bone marrow culture in patients was increased (1,684 +/- 1,151 ng/culture) compared to controls (1,087 +/- 937), but this difference was not significant (P = 0.2). However, in patients the IgA1 subclass contributed significantly (P less than 0.01) more to the IgA synthesis in culture (ratio of IgA1 over IgA: 0.96 +/- 0.02) than in controls (ratio 0.90 +/- 0.06). These findings suggest that the bone marrow may well be the site of long-term overproduction of the IgA1 found in the circulation and mesangial deposits in IgA nephropathy.

IgG subclass distribution in juvenile human tonsil: IgG3 and IgG4 results of specific antibody production using synthetic peptides.

A decapeptide with a sequence corresponding to a part of the hinge region of human IgG3 was used to prepare a mouse monoclonal antibody (Mab 330-2.2). The Mab recognized IgG3 in ELISA only when the IgG3 was denatured by mild heat. Mab specificity in immunohistochemistry was calibrated with a number of reference Mabs obtained through a WHO/IUIS collaborative study. Finally, Mab 330-2.2 was used in conjunction with a set of other isotype specific Mabs to study the (sub)class distribution of cytoplasmic Ig containing cells in palatine tonsil. IgG3 within the IgG class was strongly over-represented in our tonsil material if compared to the representation of IgG3 in serum.

Immunofluorescence: quantitative considerations.

The combination of the specificity of the antigen-antibody interaction with the sensitivity of fluorescence detection and quantitation yields one of the most widely applicable analytical tools in cell biology. Immunofluorescence (IF) signals can be measured with microscopes or flow cytometers. Choice for either of the two systems depends on the type of preparation (cells, tissues, etc.), on the type of required information (morphology, distribution, quantitation) and on the number and quality of the samples. In IF a choice has to be made for the most appropriate fluorochromes, reagents, equipment and preparative procedure, respectively, the right balance between lightsource, objective, eye pieces and filters should be sought in microscopy as well as in flow cytometry. The quantitative influence is reported of each of these variables on the eventual fluorescence intensity. New developments are appearing in IF technology. These include laser-scan microscopy, point-addressable optical sensors, phosphorescence microscopy and more sophisticated flow cytometers that allow some morphometry. The possible uses of these systems is discussed.

A Collaborative Study Comparing Three ELISA Systems for Detecting Staphylococcus aureus Enterotoxin A in Sausage Extracts.

Three ELISA's for the detection of staphylococcal enterotoxin A (SEA) were evaluated by a collaborative test in five laboratories for possible use in quality control laboratories. Two ELISA's gave quantitative results using polyclonal antibodies (PCA) or monoclonal antibodies (MCA); the third was the commercially available qualitative FEY ELISA test using PCA. Test samples comprised ripened and unripened sausage alone or spiked with 10 µg or 1 µg SEA/100 g, the latter representing a minimum emetic dose. The quantitative MCA ELISA gave more reliable and sensitive results with lower non-specific background responses, lower standard deviations and coefficients of variation than the PCA ELISA; however, the recovery of enterotoxin concentrations from extracts was lower, which was more distinct in unripened than in ripened sausage. The MCA ELISA gave lower values of absolute response but more reliable results for extracts from sausage spiked with 1 µg SEA/100 g. The FEY ELISA gave sufficient reliable results in our experiments with sausage extracts and looks adequate for controlling suspicious raw materials and food products.

The effect of divalent and univalent binding on antibody titration curves in solid-phase ELISA.

This paper describes the influence of antigen coating concentration, epitope density per antigen molecule and anti-immunoglobulin reagents on antibody titration curves in solid-phase ELISA. Based on results obtained with fluorescein as the hapten and monoclonal anti-fluorescein antibody, which were confirmed in another antigen-antibody system, it is concluded that: (a) Antibody titration curves are independent of antigen-coating concentration in a limited range of concentrations only. (b) The complex between one antibody and two epitopes ('divalent binding') is more stable than the complex between one antibody and one epitope ('univalent binding). The ratio between divalent and univalent binding depends on the epitope density per antigen molecule and on the antigen-coating concentration. (c) The prozone phenomenon can be explained by an increased instability of plate bound antibodies due to a shift from divalent to univalent binding. (d) In solid-phase ELISA a correct evaluation of the antiserum specificity can be performed only if it is ascertained that all target antigens are coated under saturating conditions.

Characteristics of IgA deposits in liver and skin of patients with liver disease.

The presence of IgA deposits in a continuous pattern along hepatic sinusoids is a specific entity for alcoholic liver disease. In superficial skin blood vessels of patients with liver disease, IgA deposits can occur. The authors characterized the deposits for IgA-subclass epitope expression and for macromolecular configuration (assessment of [hidden] J-chain determinants and of secretory component-binding capacity). A variety of monoclonal anti-IgA-subclass reagents were applied, which proved to be specific in control experiments on blastoid cells generated by pokeweed mitogen stimulation of blood mononuclear cells and frozen tissue sections of normal jejunum. IgA1 is the major component in IgA deposits in liver (n = 83) and skin (n = 31) of patients with liver disease. Macromolecular IgA is detectable in only one-fifth of the cases. The authors' data do not indicate that hepatic IgA deposits in liver disease are of gastrointestinal origin. Out of the circulating IgA pool, IgA1 appears to be most capable of being deposited in tissue.

Production and characterization of pepsin fragments of human IgA1 to determine domain-specificity of monoclonal anti-IgA antibodies.

Eight human IgA1 myeloma proteins were analysed by SDS-PAGE. These experiments showed that purified IgA1 proteins comprise both fully S-S bonded and partly S-S bonded molecules. Pepsin digestion of the IgA1 proteins yielded three four-chain and two two-chain fragments. The four-chain fragments are likely to be derived from intact IgA through cleavage of its alpha chains at different sites: between the CH2 and CH3 domains or in the hinge region. The occurrence of F(abc) (ab') fragments, with alpha chains of different lengths, showed that the alpha chains of IgA can be cleaved independently at the hinge region site. The two-chain pepsin fragments must originate from IgA molecules, which lack inter-assay-chain disulphide linkages. The fragments F(abc)2 and Fabc tended to form dimers, probably through non-covalent interactions of their CH2 domains. An immunoblotting method was used to identify Fd-, CH2- and CH3-specific anti-IgA antibodies. The CH2-specific antibodies could be subdivided into antibodies recognizing an isotype present on both four-chain and two-chain molecules or on two-chain molecules only.