Laboratory guidelines for the practical use of HIV drug resistance tests in patient follow-up.

HIV drug resistance is one of the major limitations in the successful treatment of HIV-infected patients using currently available antiretroviral combination therapies. When appropriate, drug susceptibility profiles should be taken into consideration in the choice of a specific combination therapy. Guidelines recommending resistance testing in certain circumstances have been issued. Many clinicians have access to resistance testing and will increasingly use these results in their treatment decisions. In this document, we comment on the different methods available, and the relevant issues relating to the clinical application of these tests. Specifically, the following recommendations can be made: (i) genotypic and phenotypic HIV-1 drug resistance analyses can yield complementary information for the clinician. However, insufficient information currently exists as to which approach is preferable in any particular clinical setting; (ii) when HIV-1 drug resistance testing is required, it is recommended that testing be performed on plasma samples obtained before starting, stopping or changing therapy, on samples that have a viral load above the detection limit of the resistance test; (iii) the panel recommends that genotypic and phenotypic HIV-1 drug resistance testing for clinical purposes be performed in a certified laboratory under strict quality control and quality assurance standards; and (iv) the panel recommends that resistance testing laboratories provide clinicians with resistance reports that include a list of drug-related resistance mutations (genotype) and/or a list of drug-related fold resistance values (phenotype), with interpretations of each by an experienced virologist. The interpretation of genotypic and phenotypic analysis is a complex and developing science, and in order to understand HIV-1 drug resistance reports, communication between the requesting clinician and the expert that interpreted the resistance report is recommended.

Severe accidental hypothermia: rewarming with CVVHD.

We describe 2 case reports dealing with accidental hypothermia in which continuous veno-venous haemofiltration and dialysis (CVVHD) in combination with hyperinfusion with heated crystalloids was used as a rewarming technique. In this way it also is possible to correct metabolic disturbances, even during cardiopulmonary resuscitation.

Severe hyponatremia during therapy with fluoxetine.

We described two elderly patients with severe hyponatremia that was probably caused by inappropriate secretion of antidiuretic hormone. The hyponatremia arose during fluoxetine (Prozac) antidepressant therapy.

Cyclosporin A reduces albuminuria in experimental anti-GBM nephritis independently from changes in GFR.

Cyclosporin A (CsA) can reduce proteinuria in various forms of human and experimental glomerulonephritis. This antiproteinuric effect of CsA may be the consequence of diminution of immunological damage, a drug-induced decrease of GFR, or changes in permselectivity of the glomerular basement membrane (GBM). We studied the antiproteinuric effect of CsA in the heterologous phase of a passively induced anti-GBM nephritis in the mouse. This passive model is characterized by acute exudative glomerular lesions and a dose-dependent albuminuria. Rabbit anti-mouse GBM antibodies were administered intravenously in C57B110 mice at day 4, after 3 days of pretreatment with either CsA (75 mg/kg body weight) (n = 15) or olive oil (OO, controls, n = 15) orally. CsA did not influence the severity of the histological lesions. Albuminuria was substantially reduced by CsA (CsA 1.6 +/- 1.8; OO 5.6 +/- 3.2 mg/18 h; P < 0.002). There was a considerable concomitant reduction of the GFR by CsA, as measured with a 51Cr-EDTA single-shot plasma clearance technique before (day-1) and during treatment (day 4): GFR ratio day 4/day-1 for CsA, 0.4 +/- 0.1; for OO controls, 1.1 +/- 0.6; P < 0.01. This drug-induced decrease of GFR was prevented by simultaneous treatment with phenoxybenzamine (PB) twice daily 45 micrograms orally for 4 days (GFR ratio day 4/day-1 for PB and CsA, 0.9 +/- 0.4; controls (PB and OO), 1.0 +/- 0.4; P = NS). Although the CsA-induced GFR reduction was prevented, CsA still reduced albuminuria significantly (PB and CsA, 2.2 +/- 1.8; controls (PB and OO), 5.6 +/- 1.8 mg/18 h; P = 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)

[Severe hyponatremia, possibly due to inappropriate antidiuretic hormone secretion, during use of the antidepressant fluoxetine]. / Ernstige hyponatriëmie, mogelijk door inadequate antidiuretisch-hormoonsecretie, bij gebruik van het antidepressivum fluoxetine.

In a 78-year-old female with hyponatraemia, the syndrome of inappropriate ADH secretion as a side effect of fluoxetine, a relatively new antidepressive drug, was tentatively diagnosed. In the literature 14 cases of this adverse reaction have been described. One should be careful when prescribing fluoxetine to elderly persons, notably when a diuretic is prescribed as well.

Sjögren's syndrome complicated by cryoglobulinaemia and acute renal failure.

A patient is described with Sjögren's syndrome and an IgM-kappa gammopathy. With time cryoglobulins, the cause of acute renal failure, could be detected. The kidney biopsy showed a proliferative glomerulonephritis as is frequently seen with mixed cryoglobulinaemia. Plasmapheresis and immunosuppression resulted in long-term improvement of kidney function. The differential diagnosis of acute renal failure of glomerular origin in a patient with Sjögren's syndrome is discussed. This case report illustrates once more the consequences of monoclonal B-cell lymphocyte activation in Sjögren's syndrome.

Anti-GBM nephritis in the mouse: role of granulocytes in the heterologous phase.

The role of polymorphonuclear granulocytes (PMNs) was studied in a model of anti-GBM nephritis in mice, in which PMN depletion was induced by total body irradiation of 7.5 Gy. Both in complement-normal B10.D2 new and in C5-deficient B10.D2 old mice, PMN depletion completely prevented the albuminuria occurring after injection of low doses of anti-GBM serum, and severely depressed the albuminuria after injection of high doses. In immunofluorescence, glomerular deposition of antibody and C3 was similar to that in control mice. The glomerular influx of PMNs in both the complement-normal and C5-deficient controls was inhibited to 10% or less of control values. Fibrin deposition or necrosis did not occur. Injection of F(ab')2 fragments of the anti-GBM antibody in non-irradiated mice caused only limited PMN influx and reduced the albuminuria to physiological levels, although the binding of 125I labeled F(ab')2 fragments to the glomeruli was as high as 82% of that of the complete antibody. We conclude that the albuminuria in this model is Fc-dependent and largely, if not completely, dependent on the influx of PMNs in the glomeruli. Among the many experimental models of anti-GBM nephritis, this is the first one in which the heterologous phase is complement-independent but PMN-dependent.

European multicentre evaluation of the ABBOTT Spectrum clinical chemistry analyzer.

The analytical performance of the selective multitest ABBOTT Spectrum analyser was studied according to the ECCLS guidelines and partly the CERMAB protocol in a multicentre evaluation involving laboratories from six European countries. Fifteen analytes, including the electrolytes sodium, potassium and chloride, were measured each in at least 3 laboratories, all at 37 degrees C, except the electrolytes, which are measured at room temperature. The trial lasted approximately three months and involved the collection of over 60,000 data points. It yielded the following results: 1. The precision was at least as good as the precision obtained with the comparison instruments. The majority of the coefficients of variation were between 1 and 4%. 2. The recovery for method assigned control sera values was, with few exceptions, within 10%. 3. Good agreement with respect to the method assigned values of control materials and method comparison with patient specimens to different instruments (e.g. SMAC, Hitachi 737, RA 1000) was found. 4. No drift was observed. 5. Reagent-related carry-over was not found. Specimen-related carry-over was detected in some cases, the deviation being of little or no clinical significance. 6. The manufacturer's claims regarding method linearity were as stated or exceeded. 7. The open system capability was tested and rated as very convenient. 8. The practicability of the instrument was very good.

Antiglomerular basement membrane nephritis in beige mice. Deficiency of leukocytic neutral proteinases prevents the induction of albuminuria in the heterologous phase.

Antiglomerular basement membrane (GBM) nephritis with massive albuminuria can be induced in mice by injection of heterologous antibodies against mouse GBM. The albuminuria and the glomerular lesions in this model are not mediated by complement, but are dependent on the presence of polymorphonuclear granulocytes (PMN) in the glomeruli. Neutral serine proteinases and reactive oxygen metabolites produced by activated PMN have been implicated as agents contributing to tissue damage. We examined the role of leukocytic neutral proteinases by comparing the glomerular damage and albuminuria after injection of rabbit anti-mouse GBM antibodies in normal control mice (C57BL/6J, +/+) and in beige mice (C57BL/6J,bg/bg) in which PMN are deficient of the neutral proteinases elastase and cathepsin G. The dose-dependent albuminuria that occurred in control mice after injection of 1.4-22 mg of anti-GBM antibodies was not observed in beige mice, despite a comparable influx of PMNs in the glomeruli. By electron microscopy both strains showed a similar attachment of PMN to the denuded GBM together with swelling and necrosis of endothelial cells. Elastase activity of extracts from PMN of beige mice was only 10-15% of the activity of control mice. In vitro, GBM degradation by PMN extracts of beige mice was 70% lower than that seen in control experiments. PMNs of beige and control mice showed no differences in superoxide production. In addition, administration of scavengers of reactive oxygen metabolites, such as catalase and desferrioxamine, did not prevent the albuminuria in this model. These findings support the important contribution of leukocytic neutral proteinases to the induction of albuminuria in the acute phase of anti-GBM nephritis in the mouse.

Antiglomerular basement membrane nephritis in the mouse. Study on the role of complement in the heterologous phase.

The role of complement was examined in a model of antiglomerular basement membrane nephritis in the mouse, induced by intravenous injection of goat anti-mouse glomerular basement membrane serum, and characterized by early glomerular lesions and a dose-dependent albuminuria. We compared the reaction after injection of the antiserum in complement-normal B10.D2 new mice with that in congenic, congenitally C5-deficient B10.D2 old mice, and in both strains after C3 depletion by treatment with cobra venom factor. The dose-dependent albuminuria was not affected by the absence of complement activation. Also, deficiency of C3 and/or C5 had no inhibitory effect on the histologic glomerular lesions: it did not reduce the influx of polymorphonuclear granulocytes in the glomerular capillary vessels, nor prevented the eventual intravascular coagulation. We conclude that complement-independent mechanisms are involved in the development of the heterologous phase of antiglomerular basement membrane nephritis in the mouse.

An improved sensitive and simple microassay of mouse complement.

A simple and fast hemolytic microassay was developed for the determination of classical pathway complement activity in mouse serum. The assay is based on hemolysis of sheep red blood cells (SRBC) that are sensitized with polyclonal mouse antibodies. The degree of hemolysis was measured in the reaction supernatants by photometric reading in an ELISA plate scanner at 405 nm wavelength. It was found that some batches of unpurified mouse anti-SRBC antibodies gave insufficient hemolysis. Analysis of two antibody preparations indicated that this might be caused by anti-complementary factors in the ascites fluid, and by an excess of non-complement fixing IgG1 antibodies. For optimal and standardized results, removal of anticomplementary factors and enrichment for complement fixing IgG2 antibodies was required and was achieved using protein A purified anti-SRBC IgG. In our assay it is possible to determine CH50 titers in triplicate in 80 microliters samples of individual mouse sera with high sensitivity. Using this rapid one-step method large numbers of tests could be performed in 1 day.

Post-cardiac injury syndrome and an increased humoral immune response against the major contractile proteins (actin and myosin).

To better understand the pathogenesis of the post-cardiac injury syndrome (PCIS) 2 models of cardiac injury were studied. One hundred twenty-nine patients who underwent cardiac surgery and 80 patients with acute myocardial infarction (AMI) were prospectively followed and the levels of anti-heart antibodies (AHA), anti-actin antibodies (AAA) and anti-myosin antibodies (AMA) were determined. In the surgical group, PCIS developed in 27 patients (21%) and incomplete PCIS in 36 (28%). In the AMI group, PCIS did not develop in any patient, but incomplete PCIS developed in 11 patients (14%) (p less than 0.001). The surgical group showed a significantly higher humoral immune response than the AMI group when analyzed for AHA and anti-contractile protein antibodies. After cardiac surgery, AHA developed in 59 patients (46%), AAA developed in 33 (26%) and AMA developed in 49 (38%); in the AMI group, significant levels of AHA, AAA and AMA developed in 16 (20%), 7 (9%) and 13 patients (16%), respectively. These studies show a significant correlation between the PCIS clinical classification and auto-antibodies raised against heart contractile proteins.

Detection of anti-contractile antibodies after cardiac surgery using ELISA assay.

Sera from 196 patients were collected before and after cardiac surgery to measure antibodies against heart tissue and against actin and myosin. In the post-operative period antibodies were found in 87 patients (44%) producing a cross-striated fluorescence pattern in heart tissue. Antibodies against the major contractile proteins were found in 91 patients (46%), anti-actin antibodies in 49 patients (25%) and anti-myosin antibodies in 65 patients (33%). We found a significant correlation (P less than 0.0001) between the antibodies producing a cross-striated fluorescence pattern and antibodies recognizing contractile proteins. These results suggest that contractile proteins evoke an immune response after cardiac surgery and that this response may compromise cardiac function.

Anti-GBM nephritis in the mouse: severe proteinuria in the heterologous phase.

Highly reproducible anti glomerular basement membrane (GBM) nephritis has been induced in the mouse after a single injection of rabbit or goat antibody against purified homologous GBM. The severity of albuminuria was closely related to the amount of antibody given. With doses of 4 mg or more, low serum albumin concentrations, sometimes accompanied by ascites and oedema, were observed after 1 week. Glomerular injury was characterized by an initial accumulation of polymorphonuclear granulocytes followed by thrombosis and necrosis, the extent of which defined the outcome of the glomerulonephritis. With high doses of antibody the exudative lesions entered a chronic phase, while at doses lower than 2 mg remission of the lesions occurred. Immunofluorescence studies showed prompt linear fixation of the injected antibodies to the glomerular capillary wall, accompanied by immediate binding of C3 in a fine granular pattern. Fibrin deposits appeared at 2 h in some glomeruli, increased thereafter, and were present after one day in more than 90% of the glomeruli in mice that had received 4 mg of antibody. This new reproducible model in the mouse is suited for the study of the relationship between activation of mediator systems, histological lesions, and proteinuria.