Low urine pH is associated with reduced indinavir crystalluria in indinavir-treated HIV-infected individuals.

Indinavir is a potent HIV-1 protease inhibitor included in current antiretroviral therapeutic regimens. It is associated with renal and urological complications ascribed to indinavir crystalluria. We have previously reported that indinavir crystalluria is frequently observed soon after initiation of therapy. In a cohort of 54 asymptomatic indinavir-naive HIV-1-infected individuals during their first year of treatment with indinavir, approximately 25% of urinalyses (U/A) contained indinavir crystals. Because the determinants of the crystalluria are unknown, we examined the relationship between urine specific gravity (SG) and pH, singly and in combination, and indinavir crystalluria in these subjects. A total of 579 U/A were obtained from the study subjects at their scheduled monthly outpatient medical assessments. The frequency of indinavir crystalluria was lower in U/A with lower pH, irrespective of the SG. Conversely, U/A with high pH (> or = 6.0) had a higher frequency of indinavir crystalluria, which was further influenced by the urine SG. As a result, nearly half of the U/A (46.7%) with high pH (> or = 6.0) and intermediate-high SG (> or = 1.015) contained indinavir crystals. In conclusion, the frequency of indinavir crystalluria in asymptomatic HIV-1 infected individuals during their first year of treatment with indinavir was markedly influenced by the urine pH and SG. Our findings suggest that low urine pH may have a protective effect against indinavir crystalluria across the entire range of urine SG.

Effect of chronic renal failure on the expression of erythropoietin message in a murine model.

The anemia of chronic renal failure (CRF) is largely due to decreased production of erythropoietin (EPO) by the kidney. A small amount of EPO also originates from extra-renal sources, and this would be expected to assume a more important role in maintaining erythropoiesis when renal production is impaired. In this study, we examined the production of EPO mRNA by RT-PCR in kidney, liver, and bone marrow tissues isolated from normal mice, mice rendered acutely anemic by phlebotomy, and from mice with surgically induced CRF. The induction of acute anemia results in an expected increase in the expression of EPO mRNA in renal and hepatic tissue. In contrast, while the expression of EPO mRNA was expectedly reduced in the kidney from CRF mice, it was completely absent in the liver of these same animals. EPO mRNA expression was also absent in the bone marrow in both states of acute anemia and CRF. These results show that CRF can directly or indirectly can suppress the extrarenal production of EPO by the liver and that this effect may further aggravate the anemia of CRF.

Antibody-dependent cell-mediated cytotoxicity (ADCC) assay for specific IgG antibody produced in vitro.

IgG antibody production by immune spleen cells in vitro was assayed by antibody-dependent cell-mediated cytolysis (ADCC). The use of this technique as a sensitive and reproducible alternative to indirect haemolytic plaque assays is examined.

Erythrocytosis after renal transplantation represents an abnormality of insulin-like growth factor-I and its binding proteins.

BACKGROUND: Secondary erythrocytosis is classically defined by an increase in erythropoietin (EPO) production. Despite increased levels of EPO often seen in secondary erythrocytosis, some of these forms such as that seen after renal transplantation remain undefined. Our group has recently investigated the in vivo function of insulin-like growth factor-I (IGF-I) in erythropoiesis both in humans and in a murine model of chronic renal failure. These data, and the recently recognized role of IGF-I in polycythemia vera, suggested that IGF-I might be involved in secondary erythrocytosis. METHODS: Renal transplant recipients who developed erythrocytosis after transplantation were compared to normal individuals and to renal transplant recipients without erythrocytosis. We measured fasting serum EPO and IGF-I in all three groups. Because binding proteins may modify IGF-I function, IGF-I-binding proteins (IGFBP) 1 and 3, major binding proteins of IGF-I, were also measured. RESULTS: Renal transplant recipients have significantly elevated serum of IGF-I and IGFBP3 compared to normal individuals. When transplant recipients with and without posttransplant erythrocytosis were compared, similar levels of IGF-I were found; however, the group with erythrocytosis had significantly elevated IGFBP1 and IGFBP3. No other significant differences including EPO levels were found between the groups. CONCLUSIONS: Erythrocytosis after renal transplantation represents an anomaly of both IGF-I and its major binding proteins. Further studies are under way to better define this dysregulation and determine whether IGF-I can play a more generalized role in secondary forms of erythropoiesis.

Prospective study of urinalysis abnormalities in HIV-positive individuals treated with indinavir.

Indinavir is a potent protease inhibitor widely used in combination with reverse-transcriptase inhibitors to treat human immunodeficiency virus (HIV) disease. Individuals treated with indinavir are prone to develop urinary complications, including renal colic, renal calculi, lower urinary tract symptoms, and indinavir crystalluria. Although renal stones secondary to indinavir have been described and characterized, little is known about the onset, frequency, and significance of the crystalluria. To document the longitudinal characteristics of indinavir crystalluria and associated urine abnormalities, 54 asymptomatic indinavir-naive HIV-positive individuals had urinalysis testing initially weekly and then monthly during the first year of indinavir treatment. Six hundred eight urinalyses were performed (11 +/- 2 urinalysis/subject), including 579 microscopy examinations performed by a nephrologist (10 +/- 2 examinations/subject). Baseline urinalysis results were essentially normal. After the start of treatment, indinavir crystalluria was frequently observed (67% of subjects). After the first 2 weeks, indinavir crystalluria remained constant at a frequency of approximately 25% of urine sediments examined at each test point. Other urine abnormalities, principally leukocytes (>/=10/high-power field) and casts, were observed in 39% of subjects. These abnormalities were more severe in five subjects, with concomitant increasing serum creatinine levels in three of them. Additional urine findings include the predominance of low pH (/=1.025 in 66% of urinalyses). In conclusion, abnormal urinalysis results were noted frequently during the first year of treatment with indinavir. The main findings were the high proportion of subjects with crystalluria and the relatively high frequency of crystalluria observed consistently throughout. These findings may occasionally be associated with other urine abnormalities, presumably secondary to indinavir crystalluria.

Dialyzer performance over prolonged reuse.

Studies were performed in patients on maintenance hemodialysis to evaluate the role of prolonged dialyzer reuse in the management of end-stage renal disease. For this purpose the patients were dialyzed without interruption with the same hollow fiber dialyzers (GambroR 120M) reprocessed with the Lixivitron IIR equipment. The data obtained from in vivo clearances in sixteen patients demonstrate that membrane permeability to small solutes (urea, creatinine, phosphate) is maintained up to thirty dialyzer uses. In vitro studies confirmed this observation and established that clearances of larger solutes (vitamin B12) are also maintained over similar extensive dialyzer reuse. Hematological and blood gas studies were performed serially during dialysis in five additional patients. Although circulating leucocyte and neutrophil counts, hemoglobin concentration as well as arterial pH and partial pressures of oxygen and carbon dioxide changed appropriately during dialysis, there was no observable difference from the first to the twentieth use of the same dialyzer. Thus, these results clearly demonstrate that prolonged dialyzer reuse in end-stage renal disease patients constitutes a stable form of renal replacement therapy provided adequate dialyzer reprocessing is applied.

Effect of RenNew-D as a sterilant on the performance of reused dialyzers.

RenNew-D (Alcide), a novel demand-release sporocidal agent, was employed instead of formaldehyde in the reprocessing for reuse of cuprophan hollow fiber dialyzers (Gambro) and the performance of these dialyzers was evaluated over 40 consecutive dialyses in six patients on maintenance hemodialysis. When RenNew-D was part of automated reprocessing performed with 4.3% bleach as specified by the manufacturer (Lixivitron), dialyzer survival was prolonged (16.7 +/- 7.2 uses) and hemodialysis neutropenia was unchanged with reuse. When RenNew-D was part of manual reprocessing conducted in the absence of bleach, marked improvement in dialyzer biocompatibility was observed but with a decreased survival (4.8 +/- 3.0). The majority of dialyzer failures were due to a fall in fiber bundle volume below a 85% set limit. Small solute clearances were maintained with both types of reprocessing. Dialyses were well tolerated throughout. Our data suggest that RenNew-D is a safe and efficacious product which can serve as a valuable alternative to formaldehyde for the purpose of dialyzer reuse.

Acquired cystic kidney disease: rapid progression from small to enlarged kidneys simulating adult polycystic kidney disease.

A 57-year-old man on chronic hemodialysis presented marked bilateral renal enlargement due to acquired cystic kidney disease (ACKD). He had been on hemodialysis for less than 3 years only (14 months prior to receiving a functional renal transplant which lasted 8 years, followed by 18 additional months of dialysis), before the diagnosis of ACKD was made following an episode of flank pain with gross hematuria. The marked changes in kidney appearance during this 11-year period were documented by serial ultrasound examination showing the kidneys to be of near-normal size before the start of dialysis (> or =10 cm in 1986), then shrunken and contracted 5 years later while having a functioning renal transplant (<5 cm in 1991), and markedly enlarged reaching the size of adult polycystic kidney disease after returning to dialysis (>13 cm in 1997). Since the risk of ACKD increases with duration of dialysis, we sought additional predisposing factors in this unusual case and found that 2 years after renal transplantation, the patient was diagnosed with breast cancer for which he was treated with surgical excision and tamoxifen. Based on ultrasound evidence that the tamoxifen treatment preceeded the appearance of the renal cystic changes, we wonder whether this drug may have played a role in the rapid development of ACKD.

Metabolic acidosis during urinary retention in a patient with an enterovesical fistula.

We report a patient known to have an enterovesical fistula who presented severe acute metabolic acidosis during an episode of urinary retention. The enterovesical fistula which had been intermittently symptomatic for 4 years, had developed after several intestinal surgical procedures and related intraperitoneal sepsis following resection of colon cancer 21 years previously. The patient who had a total colectomy and ileostomy, was admitted for hip replacement with the routine placement of a Foley bladder catheter. Three weeks post-operatively, the patient developed acute urinary retention following removal of the urinary catheter. The output from his ileostomy was immediately markedly increased, presumably from bladder urine diverted into the intestines through the enterovesical fistula. Within a few days he presented a normal anion gap metabolic acidosis with raised urea and stable creatinine; his clinical status deteriorated markedly with profound obtundation. These metabolic abnormalities were readily corrected by re-insertion of the Foley catheter with restoration of normal urine flow and immediate corresponding fall in the ileostomy output. Radiographic studies showed the presence of the enterovesical fistula originating from the jejunum. This is the first report of acute metabolic acidosis in association with an enterovesical fistula; the severe metabolic disturbances were triggered by the development of urinary retention resulting in the diversion of urine into the small bowel through the fistula.

Systemic amyloidosis involving the diaphragm and acute massive hydrothorax during peritoneal dialysis.

Hydrothorax secondary to trans-diaphragmatic fluid leakage through a peritoneo-pleural communication is an occasional, potentially serious complication of peritoneal dialysis. The etiology of this condition is not clear, being thought to be due either to congenital or acquired diaphragmatic fenestrations or acquired scarcity of muscle fibers in the tendinous part of the diaphragm which are compounded by increased intra-abdominal pressure during the dwell period of peritoneal dialysis. We report a 54-year-old woman who developed irreversible acute renal failure from adjuvant chemotherapy for ovarian cancer previously resected surgically. Three days after the onset of continuous ambulatory peritoneal dialysis, she developed acute respiratory distress associated with a massive right hydrothorax secondary to a peritoneo-pleural communication demonstrated by scintigraphy. At autopsy 2 weeks later, systemic amyloidosis was surprisingly found and histologic examination of the right hemidiaphragm showed the presence of amyloid, among sparse muscle fibers. This is the first case report of a distinct pathological process, i.e. amyloidosis, involving the diaphragm associated with a peritoneo-pleural communication causing massive hydrothorax at the onset of peritoneal dialysis.

Vancomycin therapy of experimental peritoneal catheter-associated infection (Staphylococcus epidermidis) in a mouse model.

Catheter-related sepsis, principally with S. epidermidis, remains a main complication of continuous ambulatory peritoneal dialysis (CAPD). A possible reason for the antibiotic resistance often displayed by these infections is the presence of bacteria growing in a protective biofilm on the catheter surface. We developed a reproducible stable model of experimental peritoneal catheter-associated infection with S. epidermidis in the mouse and used this model to examine the therapeutic efficacy of vancomycin. The response to vancomycin treatment given daily (15 mg/kg body weight) for periods from 1-14 days, relating the proportion of successful outcome (sterilization of implant infection) over time, was typical of an S-shaped biological response curve. These results extend our previous observations in vitro of the activity of vancomycin against S. epidermidis biofilm preparations and serve as a rational basis for the experimental evaluation of synergy and antagonism in the treatment of implant-associated infection.

The modulation of rifampin action against Staphylococcus epidermidis biofilms by drug additives to peritoneal dialysis solutions.

Many drugs are added to peritoneal dialysis solutions in continuous ambulatory peritoneal dialysis (CAPD) patients, principally during episodes of peritonitis when antibiotics and nonantibiotic agents are often administered together. To evaluate the influence of nonantibiotic drug additives on antibiotic action, we determined the effect of ten drug additives on the antimicrobial activity of rifampin against S. epidermidis biofilms. None of the additives had any direct effect on biofilm bacteria. While five additives did not affect rifampin action, the other five strongly antagonized rifampin. Insulin and heparin were among these five antagonists. This antagonism suggests that a significant degree of inhibition of antibiotic action may occur by the coadministration of agents in the treatment of peritonitis, particularly in diabetic patients.

The effect of peritoneal dialysis solutions on rifampin action against Staphylococcus epidermidis in the fluid and biofilm phases of growth.

Continuous ambulatory peritoneal dialysis (CAPD) catheter-associated infections can be resistant to conservative therapeutic measures despite the apparent susceptibility of the infecting bacterial strains to antibiotics. This chronicity of infection has been ascribed to the development of bacteria on the surfaces of catheter material within a protective polysaccharide shield or matrix. CAPD catheters are unique in being exposed to nonphysiological environments of dialysis fluids of varying physical properties and constitution. We determined that the rate of growth of S. epidermidis in the fluid phase in laboratory media or fresh dialysis solution was significantly influenced by pH. An acid milieu (pH 5) impaired growth compared to a neutral milieu (pH 7). Spent dialysis fluid, however, which had an approximately neutral pH, also impaired bacterial growth, the assumption being that this results from properties of spent fluid unrelated to pH. Using rifampin as the test antibiotic, we noted that its action on S. epidermidis was unaffected by pH or by the differing milieu of fresh and spent fluid. Bacterial growth was arrested regardless of the milieu. We further examined the potential of dialysis fluids to modify the interaction between rifampin and the matrix-enclosed bacteria of standardized S. epidermidis biofilms. We determined that the pH of the dialysis solution was a critical factor influencing the outcome of exposure of the biofilm to rifampin. An acid pH enhanced antibiotic activity, whereas a neutral pH was associated with antibiotic antagonism. These findings have implications in the management of S. epidermidis catheter-associated infections using rifampin.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental Staphylococcus epidermidis implant infection in the mouse. Kinetics of rifampin and vancomycin action.

Staphylococcus epidermidis implant infections remain a therapeutic challenge; they frequently result in failure of conservative management and require removal of the implant. This resistance to antibiotic therapy has been ascribed to the presence of a protective bacterial biofilm at the surface of the implant. An animal model of implant associated infection has been developed in which preformed bacterial biofilm catheter segments are implanted intraperitoneally, resulting in a chronic standardized localized infection. The authors have determined the superior rapid action of rifampin (Cieba-Geigy) compared to vancomycin hydrochloride (Eli Lilly) and determined that the combination is superior to either used alone. No rifampin resistant surviving infection was noted, which indicates the significant contribution of mammalian host defenses. This animal model is an excellent vehicle for the study of Staphylococcus epidermidis implant associated infection and the evaluation of the relative efficacy of antibiotic regimens, singly and in combination.

An assay to measure antibiotic efficacy against Staphylococcus epidermidis biofilms on implant surfaces.

Infection is a major limitation of implantable devices. Optimal antibiotic therapeutic regimes have not yet been defined. Implant-associated infections have a number of differentiating characteristics, which include the predominance of Staphylococcus epidermidis and other skin bacteria of normally low pathogenicity as the causative agents, together with a relative resistance to host defenses and to antibiotic therapy. These properties have been ascribed to the ability of the bacteria to exist on implant surfaces in the biofilm phase, which is protective. An assay of antibiotic activity using a standardized bacterial biofilm preparation of S. epidermidis is described. The assay is used to evaluate the relative efficacy of antibiotics to sterilize the biofilm, when they are used singly, or in double or triple combinations. The modulating effects of changing antibiotic concentrations and modifying the environment with CAPD variables (fresh and spent dialysis fluid, common PD solution additives) are also measured and the data summarized. It is hoped that, by using this and similar assays, individualized optimal therapeutic regimes of implant-associated infections may be logically planned.

An assay of Staphylococcus epidermidis biofilm responses to therapeutic agents.

Implant-associated infections tend to become persistent, resisting host defences and antibiotic therapy. Routine clinical laboratory testing of bacterial isolates in the fluid phase for antibiotic susceptibility may not be predictive of therapeutic outcomes and therefore a number of antibiotic regimes have been formulated empirically. The resistance of implant-associated infections has been ascribed to the production by bacteria, when adherent to the implant surface, of a shielding matrix of polymerized carbohydrates protecting the enclosed bacteria from immune defences and antibiotics. This complex of surface, bacteria and matrix is termed a biofilm. We describe a technique to evaluate the efficacy of antimicrobial agents directed against biofilm-forming strains of Staphylococcus epidermidis (a major cause of implant infections) utilizing standardized biofilm preparations formed on glass. The impact of the antimicrobial agents was assessed quantally using the end-point of permanent cessation of metabolic activity (cell death) of the entire biofilm determined by the loss of ability to reduce 2,3,5-triphenyltetrazolium chloride to a visible red intracellular precipitate of formazan. The relative rate of action of differing antimicrobial agents could be determined by the minimum period of exposure of the biofilm to the agents that is required to bring about sterilization, the clinically relevant marker. A wide range of antimicrobial substances could be evaluated, including chemical disinfectants, immunoreactive substances, antibiotics; singly and in combination; and the modifying effects of interaction with non-antibacterial therapeutic agents and other environmental factors. The technique is simple, inexpensive, reproducible and readily adaptable to the clinical situation where evaluation of therapeutic regimes for individual cases of prosthetic device-associated infection is required routinely with despatch and ease of interpretation.

A mouse model of implant-associated infection.

Infections of implanted devices are of increasing frequency and importance, representing a significant limitation of many therapeutic modalities. There are puzzling features of implant-associated infection including the changes in microbial flora, the tendency to chronicity and impaired responses to conservative modes of treatment. The concept of the bacterial biofilm as a shielding mechanism generated by bacteria adherent to artificial surfaces has recently been proposed as an explanation for these features. The biofilm is a term applied to a complex comprising the implant surface, adherent bacteria and a specialized matrix enclosing the bacteria. The matrix of the biofilm is an electrostatically charged glue-like extracellular polymer derived by bacterial enzymes acting on tissue carbohydrates, formed by bacteria when adherent to surfaces. This matrix binds the bacteria to the surface providing a sequestration affording selective protection against harmful elements of the environment, especially mechanisms of host defenses and antimicrobial agents. These biological systems are complex to study because of the dynamic interaction of the microbial variables, host defenses, properties of synthetic materials and the biofilm matrix itself. There is a need for a laboratory model in which the variables can be controlled permitting the researcher to examine the outcomes of modifying one variable at a time in a planned and orderly manner. The practical way to attain this end is the conduct of studies in a stable reproducible animal model of localized biofilm-implant infection. Staphylococcus epidermidis is a representative of the class of microorganisms predominant in implant-associated infection. This paper describes the development of a model utilizing an implant-S. epidermidis-biofilm infection localized to the peritoneal cavity of the mouse. The natural history of the infection has been well documented and is stable in all respects for periods exceeding 3 months. This chronicity is especially advantageous in analyzing the impact of long-term therapeutic modalities and necessary periods of recovery and assessment. A representative example of an experimental use of this model to determine the relative efficacy of antibiotic therapeutic regimes is described, demonstrating its scope and efficacy.

Are peritoneal host defenses really important?

Peritonitis requires a constant focus of infection plus the presence of foreign material, both amply present in the CAPD patient with an indwelling catheter and universal bacterial biofilm. Amazingly the great variety of potentially cytotoxic and immunodiluent agents with which the CAPD peritoneum is confronted on a daily basis does not produce the gross rate of sepsis that might be predicted. Clearly the profound reserve capacity of the peritoneal defense mechanisms, whose functions and interactions are poorly described and understood, contribute to the continuing success of the CAPD modality.

Antibiotic activity against Staphylococcus epidermidis biofilms in dialysis fluids.

Infection associated with medical devices may be resistant to conventional antibiotic therapy. A potential mechanism explaining this variable therapeutic response is the presence of bacterial biofilms on the surface of the prosthetic material which may confer a biologically important protective function to the entrapped bacteria. To study these questions, we prepared standardized biofilms and conventional fluid phase cultures from a panel of ten Staphylococcus epidermidis strains. All ten strains showed in the fluid phase a variable pattern of sensitivity to seven antibiotics but were uniformly sensitive to rifampin and vancomycin. In the biofilm phase they demonstrated solid antibiotic resistance with the variable exception of rifampin. The nature of the milieu was a major variable. Fresh peritoneal dialysis solution enhanced the activity of rifampin but this effect was not present with spent or neutral dialysis fluids.

Impact of dialysis fluid on the susceptibility of Staphylococcus epidermidis biofilms to rifampin.

Routine laboratory testing of the susceptibility of infecting strains of S. epidermidis to antibiotics is based on the assumptions that the infection is in contact with the stable environment of blood or tissue fluid and that the bacteria are in a susceptible phase of growth. In infections of the continuous ambulatory peritoneal dialysis (CAPD) intraperitoneal catheter, the bacteria are commonly in the resistant biofilm phase of growth, are applied to the surface of the catheter, and are exposed to the nonphysiological impact of peritoneal dialysis solutions. The possible impact of these two variables is not examined in routine testing of antibiotic susceptibility. We describe an assay of antibacterial action on a strain of S. epidermidis in the biofilm phase, exposed to a neutral control environment and to dialysis solutions, both fresh and spent. Rifampin, which has an incomplete action in a neutral control environment, was used as a sensitive indicator of possible synergistic or antagonistic interaction of the biofilm bacteria with the dialysis environments. We demonstrated that peritoneal dialysis solutions (1.5% dextrose), both fresh and spent, were synergistic with rifampin action against S. epidermidis biofilms, converting the incomplete killing action to a total bactericidal outcome, clinically essential for cure. Fresh dialysis solution was more active than spent, but in both instances the synergy was complete, indicating that the crucial complementation of the bactericidal action of rifampin was independent of pH. These findings have implications in the management of catheter-associated infections due to rifampin-susceptible strains of S. epidermidis.