Bacteriophage therapy for refractory Pseudomonas aeruginosa urinary tract infection.

We describe the success of adjunctive bacteriophage therapy for refractory Pseudomonas aeruginosa urinary tract infection in the context of bilateral ureteric stents and bladder ulceration, after repeated failure of antibiotics alone. No bacteriophage-resistant bacteria arose, and the kinetics of bacteriophage and bacteria in urine suggest self-sustaining and self-limiting infection.

Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2.

BACKGROUND: In falciparum malaria sequestration of erythrocytes containing mature forms of Plasmodium falciparum in the microvasculature of vital organs is central to pathology, but quantitation of this hidden sequestered parasite load in vivo has not previously been possible. The peripheral blood parasite count measures only the circulating, relatively non-pathogenic parasite numbers. P. falciparum releases a specific histidine-rich protein (PfHRP2) into plasma. Quantitative measurement of plasma PfHRP2 concentrations may reflect the total parasite biomass in falciparum malaria. METHODS AND FINDINGS: We measured plasma concentrations of PfHRP2, using a quantitative antigen-capture enzyme-linked immunosorbent assay, in 337 adult patients with falciparum malaria of varying severity hospitalised on the Thai-Burmese border. Based on in vitro production rates, we constructed a model to link this measure to the total parasite burden in the patient. The estimated geometric mean parasite burden was 7 x 10(11) (95% confidence interval [CI] 5.8 x 10(11) to 8.5 x 10(11)) parasites per body, and was over six times higher in severe malaria (geometric mean 1.7 x 10(12), 95% CI 1.3 x 10(12) to 2.3 x 10(12)) than in patients hospitalised without signs of severity (geometric mean 2.8 x 10(11), 95% CI 2.3 x 10(11) to 3.5 x 10(11); p < 0.001). Parasite burden was highest in patients who died (geometric mean 3.4 x 10(12), 95% CI 1.9 x 10(12) to 6.3 x 10(12); p = 0.03). The calculated number of sequestered parasites increased with disease severity and was higher in patients with late developmental stages of P. falciparum present on peripheral blood smears. Comparing model and laboratory estimates of the time of sequestration suggested that admission to hospital with uncomplicated malaria often follows schizogony-but in severe malaria is unrelated to stage of parasite development. CONCLUSION: Plasma PfHRP2 concentrations may be used to estimate the total body parasite biomass in acute falciparum malaria. Severe malaria results from extensive sequestration of parasitised erythrocytes.

Stage-dependent production and release of histidine-rich protein 2 by Plasmodium falciparum.

Because of their sequestration in the microcirculation, the pathogenic late stages of Plasmodium falciparum are under-represented in peripheral blood samples from patients with falciparum malaria. Excreted products of the parasite might help to estimate this sequestered biomass. We quantified the stage-dependent production and release per parasite of P. falciparum histidine-rich protein 2 (PfHRP2) with the objective of measuring the sequestered biomass. A simple method to relate parasite stage to parasite age was developed to facilitate this. In four isolates of P. falciparum, the median (range) PfHRP2 content was 2.0fg (0.5-4.3fg) for a young ring stage infected erythrocyte, and 5.4fg (2.1-10.2fg) for the schizont stage. The amount of PfHRP2 in the parasitized erythrocyte increased most during development to the mature trophozoite stage. The median (range) amount of PfHRP2 secreted per parasite per entire erythrocytic cycle was 5.2fg (1.1-13.0fg). A median of 89% of the total PfHRP2 was excreted at the moment of schizont rupture. This assessment of the stage-dependent release of PfHRP2 is an essential prerequisite for future studies aimed at estimating the total patient parasite mass from the peripheral blood PfHRP2 concentration.

Optimisation of an ELISA for the serodiagnosis of visceral leishmaniasis using in vitro derived promastigote antigens.

An antibody detection ELISA was developed for diagnosis of visceral leishmaniasis. Antigens released by Leishmania donovani promastigotes into a protein-free medium were used. SDS-PAGE analysis has indicated that Ld-ESM contain several protein antigens. Titration and chequer-board analyses were performed to optimise the assay protocol. Optimal results were obtained when antigen (50 microg/ml) was coated with PBS-methyl glyoxal buffer, and wells blocked with 0.5% casein. A serum dilution of 1:500 in antigen-coated wells, blocked with 0.5% casein, generated lowest absorbance with Ref-ve sera and higher absorbance with Ref+ve sera. All steps of the ELISA were performed at room temperature. The S/N ratio, the differential absorbance between the negative sample vs. the test or Ref+ve sample, was used to quantify the specific antigen and antibody reactions. An anti-human monoclonal antibody conjugated with HRP (MAb-conjugate) outperformed a commercially available anti-human polyclonal antibody conjugate (PAb-conjugate). The MAb-conjugate gave minimal background reactions with endemic sera. Optimised final assay steps mentioned below were used to evaluate sera samples from field trials. ELISA wells were coated with 50 microg/ml Ld-ESM mixed in PBS-methyl glyoxal overnight, and after removing the antigen, blocked with 0.5% casein for 1 h at RT. Patient sera along with control sera, diluted to 1:500 in PBS/T, were reacted for 1 h at RT. After washing the plate with PBS/T, wells were reacted with MAb-conjugate for 40 min at RT, and after washing, binding of antibodies was visualized by using TMB as a chromogen substrate. The relative specific binding was quantified by the S/N ratio. A batch of n=22 endemic sera from North Africa were evaluated and resulted with 100% specificity and sensitivity, 99.99% PPV and 95.45% NPV. The specificity and sensitivity of this assay will be further evaluated in planned retrospective and prospective multi-site trials.

Opportunistic infections in AIDS and their diagnosis.

AIDS patients are susceptible to a variety of infections from microorganisms as a result of their immunosuppressed condition. These infections are mainly responsible for the morbidity and mortality in these patients. The organisms responsible include protozoa, bacteria, fungi and viruses. Their presence may be a result of reactivation of latent or previous infection, or exposure to opportunistic agents. The seriousness of the patients' condition makes rapid and early diagnosis imperative so that appropriate treatment can be instituted. Methods of diagnosis including more recent technology are discussed.

Evaluation of MSA as a serum marker in breast cancer: a comparison with CEA.

In a blind study, 518 serum samples were assayed for serum levels of mammary serum antigen (MSA) by an enzyme immunoassay (EIA) using the 3E1.2 monoclonal antibody. Using 300 IU as the arbitrary cut off to distinguish normal from abnormal individuals, 75% of patients with primary Stage I carcinoma of the breast (n = 12), 89% of those with Stage II (n = 9) and 93% of those with Stage IV (n = 57) had elevated levels of MSA. A relationship was observed between the level of MSA and stage of disease, and therefore with the extent of tumour burden. Levels of MSA were also determined in a series of 19 patients undergoing chemotherapy for breast cancer. Over a 2-24 month period, the change of MSA levels corresponded with the clinical course of the disease in 17 (89%) cases. MSA levels were also raised in some patients with ovarian, colon, lung and kidney cancer, but the average level was lower than in patients with breast cancer. A comparison of CEA and MSA levels in these patients revealed that MSA was a substantially better marker for breast cancer than CEA. The results of this study demonstrate that MSA levels are elevated in patients with breast cancer and may provide a useful means of following the clinical course of patients with this disease.

The effects of oral and combined parenteral/oral immunization against an experimental Escherichia coli urinary tract infection in mice.

A double oral immunization (PO/PO) with an outer membrane protein (OMP) from a human uropathogenic strain of Escherichia coli, resulted in the partial protection of mice infected per urethrally with the same strain. Complete protection was achieved by immunizing with OMP in Freund's complete adjuvant (FCA), intramuscularly (i.m.), followed by an oral boost (i.m./PO). The PO/PO protocol stimulated mainly local urinary antibody synthesis, particularly IgA, whilst the i.m./PO regimen resulted in the appearance of both serum and urine antibodies. A single dose of OMP, 6 days after infection, rendered the mice resistant to reinfection, in contrast to non-immunized mice, and led to a significant increase in urine, serum and bile IgA anti-OMP levels. Our results confirm previous reports that the urinary tract forms part of the common mucosal immune system and provides further evidence for immunological memory in mucosal immunity. These results also demonstrate that our OMP preparation is a highly effective immunizing antigen, and that such preparations may be suitable as oral vaccines against urinary tract infection in humans.

A cell ELISA technique. The direct detection and semi-quantitation of immunoglobulin positive cells in 7 day lymphocyte cultures using the microtitre culture plates as the solid phase.

A sensitive ELISA is described which allows the detection and quantitation of immunoglobulin positive Peyer's patch and spleen lymphocytes in tissue culture microtitre plates. A methanol fixation step is used to fix the cells to the microtitre plate. The effects of various mitogens and an antigen on the number of immunoglobulin positive cells present after a 7 day culture were studied. Results were expressed either in optical densities or as percentages of the unstimulated control. The method proves a useful addition to existing techniques for studying the effects of mitogens and other agents on in vitro immunoglobulin synthesis by lymphocytes, and reflects immunoglobulin levels in the culture supernatants.

The optimum conditions for antibody production in vitro by Peyer's patch lymphocytes and a comparison between total immunoglobulins and antigen (TNP)-specific antibody synthesis following mitogen stimulation.

Using optimised conditions in terms of cell concentration (2 X 10(6)/ml), duration of culture (7 days) and pH (7.0-7.1) and the highly sensitive enzyme-linked immunosorbent assay for antibody quantitation, we assessed the effects of lipopolysaccharide (LPS) and concanavalin A (Con A) on the levels of both total immunoglobulins and TNP-specific antibodies and compared the proportion of IgA anti-TNP to total IgA synthesised following the in vivo priming of mice, 5 days prior to culture, with trinitrophenylated sheep red blood cells (TNP-SRBC). LPS stimulation resulted in an increase in total IgA (ninefold), IgM (sixfold) and IgG (eightfold) synthesis over unstimulated cultures and similar increases were seen in IgA anti-TNP (eightfold), IgM anti-TNP (fivefold) and IgG anti-TNP (twelvefold). Con A stimulation resulted in a slight increase in total IgA (threefold), IgG (twofold) but a decrease in IgM (twofold) but did not appear to affect TNP-specific antibody levels. In unstimulated cultures, IgA anti-TNP comprised 7.8% of the total IgA synthesised. In LPS and Con A stimulated cultures the proportion was 6.6% and 3.2%, respectively. Inclusion of 10% fetal calf serum in the culture medium resulted in a substantial increase in the synthesis of IgG, IgM and IgA in unstimulated cultures but increased IgA levels only in LPS stimulated cultures. Our results emphasise the importance of optimising culture conditions when studying in vitro antibody synthesis and demonstrate that Peyer's patches respond quite substantially to parenterally administered antigen and that these responses can subsequently be monitored in vitro.

A computerised ELISA system for the determination of total and antigen-specific immunoglobulins in serum and secretions.

A computerized micro-ELISA system is described which features rapid and simple procedures for sample storage and transfer, plate washing and calculation of results. Calculations are performed by a Commodore Pet desk computer interfaced with a Titertek Multiskan micro-ELISA plate reader. Up to 1200 analyses per day can be performed by one person. Its application to the measurement of total immunoglobulin isotype and class specific antibody to parainfluenza type 1 (Sendai virus) in mice is described.

Identification of lactoferrin as the granulocyte-derived inhibitor of colony-stimulating activity production.

Lactoferrin (LF), the iron-binding protein present in the specific granules of mature granulocytes has been identified as colony inhibitory factor (CIF) which suppresses granulocyte--macrophage colony stimulating activity (CSA) production by monocytes and macrophages in vitro and rebound granulopoiesis in vivo. Separation of LF and CIF by isoelectric focusing confirmed that the regions of inhibitory activity corresponded in both to a pH of congruent to 6.5. In addition, the purified immunoglobulin fraction of rabbit anti-human LF antiserum, but not rabbit anti-transferrin (TF), inactivated the capacity of LF and CIF to inhibit CSA production, an effect blocked by prior incubation of anti-LF with neutralizing concentrations of LF. Suppression of CSA production correlated with the iron-saturation of LF; APO-LF (depleted of iron) was only active concentrations greater than 10(-7) M, native LF (8% iron saturated) was active at 10(-15) M, and fully iron-saturated LF inhibited at 10(-17) M. Suppression of CSA production occurred within a 1/2-h preincubation period with human blood monocytes but was reversed by bacterial lipopolysaccharide (LPS). This reversal was dependent on the relative concentrations of LF to LPS. Serum TF, a biochemically similar iron-binding protein which is antigenically distinct from LF, was only minimally active at concentrations greater than 10(-6) M. LF did not inhibit exogenously stimulated human granylocyte and macrophage colony-forming cells or erythropoietin-dependent human or murine erythroid colony- or erythroid burst-forming cells. Microgram quantities of LF acted in vivo to inhibit rebound granulopoiesis and CSA production in CD1 and C57Bl/6 mice pretreated with cyclophosphamide. These results strongly implicate LF as a physiological regulator of granulopoiesis.

Suggested models of ecotaxopathy in lymphoreticular malignancy. A role for iron-binding proteins in the control of lymphoid cell migration.

In the present paper we apply the "ecotaxis hypothesis" to the analysis of lymphocyte distribution in Hodgkin's disease and other forms of lymphoid malignancy. The results lead us to consider the possiblity that metal-binding proteins, namely ferritin, transferrin and lactoferrin, play a role in lymphocyte ecotaxopahty. It is suggested that in Hodgkin's disease, a failure of lymph node and spleen monocytes to handle iron normally could explain most of the hematologic, immunologic, pathologic, and epidemiologic features of the disease.

A combined autoradiography--immunofluorescence technique for the study of lymphocyte traffic in relation to antigen localisation.

A combined autoradiography-immunofluorescence method is described for the simultaneous detection of antigens and lymphoid cells on cryostat tissue sections. The method is discussed here with reference to studies on cell traffic and antigen localisation in the chicken spleen.