Characteristics of enzyme-linked immunosorbent assay for detection of IgG antibodies specific to Сhlamydia trachomatis heat shock protein (HSP-60)

The goal of this work was to study sensitivity and specificity of the developed ELISA set for the identification of IgG antibodies against Chlamydia trachomatis HSP-60 (using biotinylated tyramine-based signal amplification system). The study was conducted using a panel of characterized sera, as well as two reference ELISA sets of similar purpose. According to the results of ELISA informative value parameters, the ELISA we have developed showed the highest specificity and sensitivity parameters (no false negative or false positive results were registered). In 4 out of 15 intralaboratory panel serum samples initially identified as negative, anti-HSP-60 IgG-antibodies test result in reference ELISA sets upon dilution changed from negative to positive. The nature of titration curves of false negative sera and commercial monoclonal antibodies А57-В9 against C. trachomatis HSP-60 after incubation for 24 h was indicative of the presence of anti-idiotypic antibodies in these samples. Upon sera dilution, idiotypic-anti-idiotypic complexes dissociated, which caused the change of test result. High informative value of the developed ELISA set for identification of IgG antibodies against C. trachomatis HSP-60 has been proven. Anti-idiotypic antibodies possessing C. trachomatis anti-HSP-60 activity and being one of the causes of false negative results of the relevant ELISA-based tests have been identified in blood sera of individuals infected with chlamydial genitourinary infection agents.

De novo once-monthly darbepoetin alpha treatment for the anemia of chronic kidney disease using a computerized algorithmic approach.

BACKGROUND: Anemia of chronic kidney disease (CKD) has been traditionally treated by erythropoiesis-stimulating agents (ESAs) and/or iron following manual determination of dose. We hypothesized that once-monthly (QM) algorithmically dosed darbepoetin α (DA) and iron administration would successfully treat anemia of CKD in ESA-naïve CKD subjects. METHODS: QM DA and iron doses were determined via a computerized program targeting a hemoglobin (Hb) of 10.5 - 12.5 g/dl in anemic, ESA-naïve, CKD Stages 3 - 5 subjects. Six consecutive QM doses were administered. Hb, ferritin, and transferrin saturation were recorded. Data are presented as means ± standard deviation. RESULTS: Anemia was identified in 133 subjects, with a mean follow-up of 188 days. DA doses and Hb were significantly greater at Months 3 and 6 compared to baseline (p < 0.05); DA doses were 109 ± 68 µg and 118 ± 91, respectively, at Months 3 and 6. Hemoglobin levels were correspondingly 11.3 ± 1.1 g/dl and 11.3 ± 1.0. 78% of patients achieved the target Hb by 6 months of therapy. The elevation of Hb was greater in non-proteinuric than proteinuric subjects at 6 months of treatment (11.6 ± 0.8 g/dl vs. 11.0 ± 1.1; p < 0.05), despite lower DA dose (96 ± 76 µg vs. 139 ± 98; p < 0.05). CONCLUSION: Successful treatment of the anemia of CKD by QM DA based upon a computerized dosing program was achieved by 6 months in 78% of ESA-naïve, CKD subjects.

The native arteriovenous fistula in 2007. Surveillance and monitoring.

In the past 5 years, some clinical trials have questioned the value of surveillance in managing vascular accesses. Although prolongation of access life span is an important end point, reduction of thrombotic events reduces patient risks resulting from loss of access patency. Most of the available evidence suggests that detection of stenosis and prevention of thrombosis is valuable. When a test indicates the likely presence of a stenosis, then venography or fistulography should be used to definitively establish the presence and degree of the stenosis. In most but not all cases, angioplasty should be performed if the stenosis is greater than 50% by diameter. The value of routine use of any surveillance technique for detecting anatomic stenosis alone, without concomitant functional assessment by measurement of access flow, venous pressure, recirculation or other physiologic parameters, has not been established. Stenotic lesions should not be repaired merely because they are present. If such correction is performed, then intraprocedural or periprocedural measurement of access flow (QA) or intra-access pressure should be conducted to demonstrate a functional improvement with a successful percutaneous transluminal angioplasty.

The native arteriovenous fistula in 2007. Research needs.

Several research questions are open in the field of vascular access for hemodialysis. The present paper reviews both prognostic issues, such as the identification of factors for patient stratification before access insertion, and intervention questions, such as comparison of the advantages and disadvantages of different surgical solutions, the effects of different medications on vascular pathology, the different cannulation practices to prevent vessel wall lesions and technologies for early diagnosis of access dysfunction. Given that the quality of the available literature in nephrology is often suboptimal, nephrologists need to pay special attention to methodology issues before embarking on expensive multicenter studies.

The continuous erythropoietin receptor activator (C.E.R.A.) corrects anemia at extended administration intervals in patients with chronic kidney disease not on dialysis: results of a phase II study.

AIM: This study was designed to assess the potential of the continuous erythropoietin receptor activator (C.E.R.A.) to correct anemia at extended administration intervals in erythropoiesis-stimulating agent-naive patients with chronic kidney disease (CKD) not on dialysis and to determine its optimal starting dose. METHODS: Patients were assigned to subcutaneous C.E.R.A. at 3 doses: 0.15, 0.30 and 0.60 microg/kg/wk. During the first 6 weeks, dose adjustments for efficacy were not permitted in order to assess dose response. Within each of the 3 dose groups, patients were randomized to receive C.E.R.A. QW, Q2W or Q3W; the total dose during the first 6 weeks was the same for a particular dose group across the frequency subgroups. During the next 12 weeks, dose was adjusted according to predefined hemoglobin (Hb) criteria. The primary efficacy parameter was change in Hb over 6 weeks, estimated from regression analysis between baseline and the point at which the patient received a dose change or blood transfusion. It therefore provided an estimate of Hb increase based on starting dose. Other endpoints included Hb response rate (proportion of patients with a Hb increase > 1.0 g/dl on 2 consecutive occasions). A 1-year extension period investigated long term tolerability and efficacy. RESULTS: A dose-dependent relationship was noted in the mean change in Hb from baseline over 6 weeks (p < 0.0001), independent of administration schedule (p = 0.9201). There was also a significant relationship between Hb change and median serum C.E.R.A. concentration (p < 0.0001). Erythropoietic responses were sustained in all groups with mean changes from baseline in Hb > 1.2 g/dl observed at doses > or = 0.30 microg/kg/wk. Hb response rate increased with increasing dose: 67, 72 and 90% with C.E.R.A. 0.15, 0.30 and 0.60 microg/kg/wk, respectively. Generally, the median Hb response time was faster with increasing dose (89, 43 and 31 days, respectively). Response was unrelated to administration frequency. Stable Hb concentrations were maintained throughout the 1-year extension period. C.E.R.A. was generally well tolerated, and the most common adverse events were hypertension, urinary tract infection and renal failure. CONCLUSIONS: C.E.R.A. corrected anemia and maintained sustained and stable control of Hb over 1 year. These results suggest that 0.60 microg/kg subcutaneous C.E.R.A. given twice monthly is a suitable starting dose for further investigation in Phase III studies in patients with CKD not on dialysis.

Resolving the paradigm crisis in intravenous iron and erythropoietin management.

Despite the proven benefits of intravenous (i.v.) iron therapy in anemia management, it remains underutilized in the hemodialysis population. Although overall i.v. iron usage continues to increase slowly, monthly usage statistics compiled by the US Renal Data System suggest that clinicians are not implementing continued dosing regimens following repletion of iron stores. Continued therapy with i.v. iron represents a key opportunity to improve patient outcomes and increase the efficiency of anemia treatment. Regular administration of low doses of i.v. iron prevents the recurrence of iron deficiency, enhances response to recombinant human erythropoietin therapy, minimizes fluctuation of hemoglobin levels, hematocrit levels, and iron stores, and may reduce overall costs of care. This article reviews the importance of i.v. iron dosing on a regular basis in the hemodialysis patient with iron-deficiency anemia and explores reasons why some clinicians may still be reluctant to employ these protocols in the hemodialysis setting.

Effects of systemic hemodynamics on flow within vascular accesses used for hemodialysis.

Absolute value of access flow (QA) and change in flow (deltaQA) over time are major determinants of access patency. However, QA may change in response to variation in systemic hemodynamics among dialysis sessions. We examined the effect of mean arterial pressure (MAP), cardiac output (CO), and segmental resistances (R) on QA. Access flow and CO (L/min) were determined by Transonic ultrasound dilution. Static intra-access pressures (mm Hg) at the arterial segment (AS) and venous segment (VS) were determined with the access unoccluded. During access occlusion (O), the AS pressure was equated to arterial pressure (MAPo), whereas the VS pressure reflected venous pressure (VP). Total and segmental vascular resistances (mm Hg-min/L) were calculated as deltaP/Q. We studied 58 arteriovenous (AV) grafts and 35 autologous AV fistulae (AVF) with measurements on two or more occasions in 43 grafts and 25 AVF. MAPC differed from MAPo by >20 mm Hg in 22% of patients. AS (58 +/- 2 vs. 31 +/- 2) and VS (40 +/- 1 vs. 25 +/- 2) were greater in grafts than in AVF, whereas VP was equal. Access flow (0.91 +/- 0.03 vs. 0.91 +/- 0.05 L/min), cardiac output (5.1 +/- 0.1 vs. 5.5 +/- 0.2 L/min), and total access resistance (115 +/- 5 vs. 11 +/- 6) were equal in grafts and AVF, but non-access systemic R was lower in patients with AVF that those with grafts (26 +/- 1 vs. 30 +/- 1). AS and VS resistances were greater in AVF than grafts (87 +/- 6 vs. 54 +/- 3 and 37 +/- 3 vs. 16 +/- 3). Multivariate analysis indicated that CO and ipsilateral MAPo affected flow in both access types. In grafts, all three access resistance elements, AS, VS, and total independently influenced flow, whereas in AVF, the VS did not. Unexpectedly, the ratio of systemic to access resistance also influenced access flow. The pressure in the venous system draining the access affected access flow in AVF but not grafts. We conclude that the hemodynamics of grafts and AVF differ. Cardiac output, MAP, and the arterial segment resistance influence QA in both access types and need to be considered when evaluating QA as part of the trend analysis for detecting access dysfunction.

Maintaining higher TSATs and other iron indices is beneficial in management of anemic hemodialysis patients.

The National Kidney Foundation Kidney Disease Outcomes Quality Initiative and other groups support maintaining transferrin saturation (TSAT) levels above 20% and serum ferritin levels above 100 ng/ml to ensure adequate erythropoiesis in hemodialysis patients receiving erythropoietin. However, researchers have found that even patients with TSATs above 20% may still have functional iron deficiency. This article presents information that supports the fact that maintenance of TSATs between 30% and 50%, through the use of continuous intravenous iron therapy, results in improvement of anemia, reduction in erythropoietin dose requirements, and an increase in the reticulocyte hemoglobin content. The implications of these findings for clinical practice are also discussed.

Unraveling the realities of vascular access: the Network 11 experience.

Arteriovenous (AV) fistulae are well recognized as the preferred vascular access for hemodialysis, yet national data show that only 23% of patients used an AV fistula in 1997. To identify barriers to the placement of native AV fistulae, the Renal Network of the Upper Midwest, Inc. (End-Stage Renal Disease [ESRD] Network 11) initiated a vascular access project to look at the process of referral for patients beginning hemodialysis in the first 6 months of 1999. Of these patients, 63% began hemodialysis with a catheter as the only access, 22% had an AV fistula placed (but only 14% used an AV fistula for their first dialysis treatment), and 15% began with a graft. About 40% of patients were referred to a nephrologist less than 1 month before dialysis, allowing little chance for permanent access placement. Yet 27% of patients used a catheter on the first hemodialysis treatment and were seen by a nephrologist more than 1 month before starting dialysis, indicating the presence of an opportunity to improve. At 6 months after initiation of dialysis, 25% of patients who began dialysis using a catheter were using an AV fistula and 35% were using a graft. Network 11 plans to use this information to promote early referral of patients to a nephrologist and subsequent prompt referral of such patients to a vascular surgeon. Other activities to improve vascular access management are also indicated.

Physiological and pharmacodynamic considerations for route of EPO administration.

Three intertwined issues--effectiveness, dosage, and route of administration--dominate discussion about recombinant human erythropoietin (rHuEPO). The major biological effect of rHuEPO is to regulate the number of committed erythroid precursors and to cause them to mature into erythrocytes. The constant presence of rHuEPO is critical to the sustenance, multiplication, and differentiation of committed erythroid progenitors that otherwise undergo apoptosis and die before they reach maturity. The route for rHuEPO administration influences the plasma concentration-time profiles. The erythropoietic response is not dependent on the peak concentration of rHuEPO achieved but on the duration of time that rHuEPO levels are maintained above a critical concentration. High levels immediately after intravenous doses are unnecessary to either induce or to sustain erythropoiesis. During the period of relative rHuEPO deficiency that invariably follows intravenous administration, committed but still rHuEPO-dependent cells undergo apoptosis and die in the bone marrow. The subcutaneous route sustains rHuEPO levels above basal levels in the interdialytic period, prevents death of rHuEPO-dependent cells, and results in more efficient and more sustained erythropoiesis. Areas under active investigation include modifications of the parent hormone and novel delivery systems that decrease elimination and maximize the residence time of rHuEPO in the circulation.

Defining a renal anemia management period.

Estimates of the prevalence of anemia during chronic renal insufficiency (CRI) vary depending on how anemia is defined. An analysis of patients beginning dialysis in the United States found that 67% had a hematocrit of less than 30% and 51% had a hematocrit of less than 28%. The anemia of CRI, therefore, appears to be prevalent even as it is underrecognized and undertreated-despite the widespread realization that there is much to be gained by treatment with recombinant human erythropoietin, with little risk of accelerating the progression of kidney disease. It is difficult to separate the effects of anemia in CRI from those of other comorbid conditions, but it is clear that anemia is a strong predictor of mortality and cardiac morbidity. Correction of anemia would be expected to negate the contribution of anemia to the mortality and cardiac morbidity associated with CRI. While this hypothesis is well-validated in hemodialysis patients, data in the population with CRI are preliminary but encouraging. Recent small prospective studies have established that treatment of anemia with recombinant human erythropoietin can reverse some degree of the cardiac morphological changes seen in CRI. While awaiting the results of large long-term clinical trials, the concept of the renal anemia management period (RAMP) draws attention and focus to the need for proactive and aggressive treatment of anemia among patients with CRI. The RAMP is defined as the period of time after the onset of CRI during which anemia develops, requiring diagnosis and treatment. Treatment of anemia during the RAMP has the potential to ameliorate, or even prevent, significant future morbidity in patients with CRI.

Iron and cardiac disease in the end-stage renal disease setting.

Erythropoietin (EPO) therapy and appropriate iron administration are important aspects for managing the anemia of end-stage renal disease (ESRD). Achieving target hemoglobin levels of 11 to 12 g/dL and optimizing iron balance should improve clinical outcomes and increase patient quality of life. However, concerns have been raised about parenteral iron supplementation leading to excessively high iron levels, which may induce increased oxidative stress and risk for cardiovascular disease. Increased oxidative stress is often already present in patients with chronic renal disease and in patients with ESRD undergoing hemodialysis. The "iron hypothesis" proposes that excess iron is associated with increased risk for cardiac disease. While some studies have found an association between high iron levels or increased iron consumption with elevated risk for cardiac disease in subjects without renal disease, others have not found this association. Indeed, several studies suggest that achievement of target hematocrit levels in ESRD patients improves several clinical outcomes and that anemia itself is a risk factor for cardiac disease. Well-designed prospective studies are needed before the relationship between supplemental iron administration, excess iron, and cardiac disease can be firmly established.

An indistinct balance: the safety and efficacy of parenteral iron therapy.

Recombinant epoetin therapy and correction of the chronic anemia of renal failure have greatly reduced the number of red cell transfusions and hence the propensity to iron overload. The majority of HD patients require intravenous iron therapy to achieve the hematocrit levels that correspond to improved outcome measures. Although the short-term benefits of intravenous iron have been clearly defined, the long-term risks of intravenous iron are less well-defined. Iron overload before the availability of epoetin constituted a serious problem; our review of the literature does not decisively conclude that these patients had more serious bacterial infections or increased mortality when compared with their non-iron overloaded counterparts, unless chronic transfusion-related hepatic disease was superimposed. Specifically, no data unequivocally confirm that iron overload from parenteral iron contributes to all-cause patient morbidity or mortality. Furthermore, therapy that maintains intravenous iron optimal iron stores and replaces iron losses associated with the dialytic procedure does not engender iron overload in the carefully monitored patient. Optimized anemia therapy in ESRD requires individualized and specific application of epoetin and iron for each patient, and significant cost savings can result from such a strategy. Prospective studies are clearly necessary to define those parameters that reflect adequacy of iron storage in renal failure patients. We should develop alternative means of iron delivery and develop monitors that accurately discriminate between patients who will respond to additional iron therapy and those who will not. Whether ferritin should be supplanted by another parameter and whether iron itself poses an increased risk to those patients it has so beneficially served are issues that must be resolved. Until these answers are known, the importance of carefully crafted iron therapy cannot be overstated.