Comparison and application of a novel genotyping method, semiautomated primer-specific and mispair extension analysis, and four other genotyping assays for detection of hepatitis C virus mixed-genotype infections.

To date the true prevalence of hepatitis C virus (HCV) mixed-genotype infections has not been established mainly because currently available methods are not suitable for the detection of mixed genotypes in a viral population. A novel semiautomated genotyping method, primer-specific and mispair extension analysis (S-PSMEA), which is more reliable than other genotyping assays was developed for detection of HCV mixed-genotype infections. A genotype present at levels as low as 0.8% in a defined mix of HCV genotypes was detected, showing a 20-fold increase in sensitivity over that of direct DNA sequencing. A total of 434 HCV isolates were genotyped and analyzed for a comparative study of the accuracy between S-PSMEA and four current genotyping methods. The results showed that viruses in approximately 40% of the samples from this group determined to be infected with mixed genotypes by S-PSMEA were undetected by direct DNA sequencing due to its low sensitivity. Type-specific PCR, line probe assay, and restriction fragment length polymorphism analysis performed poorly, being able to identify only 38.5, 16.1, and 15.4% of mixed-genotype infections, respectively, that were detected by direct DNA sequencing. The prevalence of mixed-genotype infections detected by S-PSMEA was 7.9% (12 of 152 donors) among HCV-infected blood donors, 14.3% (15 of 105) among patients with chronic hepatitis C, and 17.1% (6 of 36) among thalassemia patients who had received multiple transfusions. The data lead us to conclude that HCV mixed-genotype infections are more common than previously estimated and that S-PSMEA may be the method of choice when detection of genotypes present at low levels in mixed-genotype infections is required due to its higher level of sensitivity.

Effective treatment of secondary hyperparathyroidism in hemodialysis patients by titration of intravenous calcitriol dosage.

AIM: Effective treatment of secondary hyperparathyroidism (HPTH) with intravenous (i.v.) administration of calcitriol in hemodialysis patients. PATIENTS AND METHODS: The current study evaluates the use of i.v. calcitriol dosing in relation to the severity of the HPTH in 35 hemodialysis patients with serum phosphate < 6.5 mg/dl. Arbitrarily, patients with plasma IPTH levels (intact PTH) between 288 and 576 pg/ml (288 pg/ml = four-fold the upper normal limit) were given initially 1 microg i.v. calcitriol at the end of each dialysis (group A, n = 15). Patients with IPTH between 577 and 864 pg/ml received 2 microg i.v. calcitriol (group B, n = 10) and patients with IPTH more than 865 pg/ml were given 3 - 4 microg i.v. calcitriol (group C, n = 10). As IPTH levels decreased, the dose of i.v. calcitriol was also decreased gradually. Patients were followed-up for 4 months after the end of calcitriol treatment. RESULTS: During the i.v. calcitriol treatment period, the observed plasma IPTH concentrations compared with the baseline values were significantly lower (p < 0.01 for A and B group and p < 0.05 for C group) from the sixth month onwards in group A and C and from the third month onwards in group B. At the 12th month of follow-up, all patients being off i.v. calcitriol treatment for four months, a sharp and significant increase (p < 0.01 for group A and B and p < 0.05 for group C) of plasma IPTH was recorded in all three groups of patients. Alkaline phosphatase was also gradually decreased in all studied groups. Serum Ca and P remained unchanged in most patients. CONCLUSION: In conclusion, the study presented here demonstrates that the titration of i.v. calcitriol dosage according to the severity of HPTH is an effective and safe treatment of HPTH in chronic hemodialysis patients. It also shows that parathyroidectomy could be avoided in the majority of patients with severe HPTH, if an appropriate dose of calcitriol not aggravating hyperphosphatemia is administered.

Survival and complications of 225 catheters used in continuous ambulatory peritoneal dialysis: one-center experience in Northern Greece.

This study reports our experience with permanent peritoneal catheters. From July 1983 until December 1997, 225 catheters were implanted surgically in 207 patients (120 males, 87 females) with mean age of 58+/-16 years (range: 2-82 years), and a mean duration of continuous peritoneal dialysis (CAPD) of 21.9+/-21.3 months (range: 1-145 months). Two hundred and seventeen catheters were used in 199 patients suffering from end-stage renal disease (ESRD), and 8 catheters in 8 patients with end-stage heart failure resistant to medical therapy. One patient used 3 catheters and 16 patients used 2 catheters. The catheters used were: Tenckhoff, 2; Oreopoulos-Zellerman-1 (OZ-1), 10; OZ-2, 205; and OZ-pediatric, 8. All catheters were implanted by the same surgical team, through a paramedian incision under local anesthesia. By life table analysis, the actuarial survival rates at 1 year, 2 years, 3 years, and 5 years were 97%, 92%, 87%, and 82% respectively for all catheters. The catheter-related complications were: 5 obstructions, 2 dislodgments, 13 dialysate leaks (6 early; 7 late), 90 exit-site/tunnel infections (in 56 patients), 2 cuff extrusions, and 37 hernias (in 31 patients). Eighteen catheters were replaced for persistent peritonitis (15 cases), dislodgment (1 case), obstruction (1 case), and accidental shortening (1 case). The total observation period was 4526 patient-months. The overall incidence of peritonitis was one episode per 15 patient-months, and of exit-site/tunnel infections was one episode per 50 patient-months, with a significant improvement during the last years. We conclude that OZ catheters implanted surgically through a paramedian incision have a very high survival rate and a low complication rate.

Estimating urea clearance in patients on continuous ambulatory peritoneal dialysis: a multivariate analysis.

The purpose of this study was to determine if Kt/V urea in continuous ambulatory peritoneal dialysis (CAPD) could be estimated by a multivariate model based upon simple clinical observations. The study included 439 clearance studies in 301 CAPD patients followed in 8 dialysis centers. Weekly urea clearance, 24 h urine volume and 24 h drain volume were normalized to body water by the formulae of Watson (Kt/V, UV/V and DV/V respectively). Adequate dialysis was defined as Kt/V > or = 2.0 weekly. Subjects at 2 units were used to derive the models, while others were used for model validation. Stepwise multiple linear regression was performed on the derivation set (DS) to identify the clinical variables that correlated with Kt/V. The model was then used to estimate Kt/V for the validation set (VS). In the DS, 110 clearance studies were performed in subjects with residual renal function. Multiple linear regression showed that weekly Kt/V was defined by the expression: Kt/V=1.48 + 24.1 (UV/V) + 2.92(DV/V) - 0.049 (serum creatinine) (r=0.750, p<0.001). In 204 VS studies, the correlation between estimated and measured Kt/V was 0.633. There were marked differences in the proportion of adequately dialyzed patients when Kt/V estimated from the formula shown was <2.0, between 2.0 and 2.3, and >2.3 weekly (7.9%, 54.7% and 79.7%, respectively; p2.3 weekly (8.1%, 68.8%, and 100%, respectively; p<0.001). The risk of low Kt/V can be estimated by multivariate linear models requiring only simple clinical measurements.

Primer specific and mispair extension analysis (PSMEA) as a simple approach to fast genotyping.

A simple method, primer specific and mispair extension analysis (PSMEA) with pfu DNA polymerase was developed for genotyping. PSMEA is based on the unique properties of 3'-->5' exonuclease proofreading activity. In the presence of an incomplete set of dNTPs, pfu was found to be extremely discriminative in nucleotide incorporation and proofreading at the initiation step of DNA synthesis, completely preventing primer extension when mispair(s) are found adjacent to the 3'-end of the primer. This has allowed us to accurately detect nucleotide variations, deletions and insertions for fast genotyping.

Histamine and serotonin in uremic pruritus: effect of ondansetron in CAPD-pruritic patients.

Pruritus is a common, unpleasant symptom of uremic patients. Serotonin and histamine have been reported as possible mediators ofuremic pruritus, and ondansetron is a potent and selective inhibitor of 5-HT3 receptors. The aims of our study were (1) to evaluate the effect of ondansetron on uremic pruritus in continuous ambulatory peritoneal dialysis (CAPD) patients and its safety and (2) to investigate the role of histamine and serotonin in uremic pruritus. To study the prevalence and pathogenesis of uremic pruritus, CAPD and hemodialysis (HD) patients were asked to complete a pruritus questionnaire. The replies were scored based on numerical scales, and the results were evaluated by the same investigator who did not know the patients. Pruritus was graded, according to the total points for each patient, as mild, moderate, or severe. Of 54 patients on HD, 29 (53.7%) had pruritus, and of 43 patients on CAPD, pruritus was present in 21 (48.8%). In HD patients, pruritus was mild in 14 (48.3%), moderate in 12 (41.4%), and severe in 3 (10.3%) patients; the distribution in CAPD patients was 9 (42.9%), 10 (47.6%), and 2 (9.5%), respectively. There was no correlation between the presence and severity of pruritus and age, sex, primary renal disease, duration of dialysis, dialysis solutions used, and hematological and biochemical parameters except for serum histamine and serotonin levels and their product. Plasma histamine levels in CAPD patients were 13.1 +/- 1.1 ng/ml in pruritic and 11.0 +/- 3.9 ng/ml in nonpruritic patients (p = 0.06), serum serotonin levels were 115.6 +/- 43.3 ng/ml and 64 +/- 42.3 ng/ml (p < 0.05), respectively, and the histamine x serotonin product was 1,461 +/- 576 and 646 +/- 545 (p < 0.01), respectively. Eleven CAPD patients (6 males, 5 females) with a mean age of 66 (range 33-83) years and an average time on CAPD of 18 (range 3-31) months with moderate to severe pruritus were treated with ondansetron (4 mg twice daily p.o.) for a mean period of 3 (range 1-5) months. All patients responded to the treatment. There was a significant reduction of the severity of pruritus from the start of treatment, and on the 3rd day the pruritic score (mean value) was 10 (range 5-19) points, while at time 0 (before treatment) it was 26 (range 19-37) points (p < 0.0001). Pruritus disappeared in 7 patients at the end of the 1st week and in all patients at the end of the 2nd week of treatment. This effect was maintained during the study. Plasma histamine levels decreased significantly during the treatment from 12.9 +/- 1.2 to 6.7 +/- 5.9 ng/ml (p < 0.05). Also, serum serotonin levels were reduced from 125.1 +/- 47.8 to 59.3 +/- 27.5 ng/ml (p < 0.05) at the end of the 1st month of treatment, and the histamine x serotonin product showed a more significant reduction: from 1,544 +/- 656 to 454 +/- 436 (p < 0.01). Three patients reported an improvement in their nausea and vomiting during the treatment. Weekly clinical and laboratory examinations showed no side effects, adverse reactions, or other complications. Our data indicate that ondansetron is an effective, safe, and well-tolerated drug for the treatment of uremic pruritus in CAPD patients and that histamine and serotonin may have a crucial role in the appearance or perception of the uremic pruritus.

Serum albumin in continuous peritoneal dialysis: absence of universal predictors.

Factors shown to affect serum albumin concentration in continuous peritoneal dialysis (CPD) were compared between two CPD populations residing in Greece (patient n = 108) and the United States (patient n = 194). Compared to the U.S. group, the Greek CPD population had higher serum albumin levels (35.1 +/- 4.6 vs 33.9 +/- 5.0 g/L, p = 0.031), was older (61.2 +/- 12.0 vs 52.7 +/- 16.5 years, p < 0.001), and had a greater number of high or high-average peritoneal solute transport types (69.4% vs 52.1%, p = 0.003). The American CPD population had a higher number of diabetics (53.1% vs 27.8%, p < 0.001), higher total Kt/Vurea (2.06 +/- 0.57 vs 1.93 +/- 0.46 weekly, p = 0.046), and higher total creatinine clearance (76.3 +/- 38.7 vs 63.4 +/- 23.5 L/1.73 m2 weekly, p < 0.001), while normalized protein nitrogen appearance values were comparable (0.95 +/- 0.21 in the Greeks vs 0.94 +/- 0.22 g/(kg x 24 hr) in the Americans, NS). A logistic regression model developed in the United States identified advanced age, diabetes, and high/high-average peritoneal solute transport as the predictors of hypoalbuminemia (serum albumin < 35 g/L). This model generated the following areas with 95% confidence intervals (CI) under the receiver operating characteristic (ROC) curve: in the Greek CPD population, ROC area 0.594 (95% CI 0.486-0.702); in the American CPD population, ROC area 0.850 (95% CI 0.810-0.890). In Greek CPD patients serum albumin appears to be affected by factors other than those identified in North America. This complicates comparisons of serum albumin, and probably morbidity and mortality, between CPD populations residing in different parts of the world.

The relationship between urine volume and drain volume as determinants of fractional urea clearance in continuous peritoneal dialysis.

A logistic regression model developed and validated in Albuquerque identified 24 hr urine volume (UV) and 24 hr drain volume/(body water) (DV/V) as predictors of fractional urea clearance (KT/V) in continuous peritoneal dialysis (CPD). Solution of this model provided DV/V values consistent with KT/V at least equal to a target KT/V for a given UV. The predictive accuracy of these unique DV/V values was tested in urea kinetic studies performed in 108 CPD patients in Thessaloniki and Athens with UV varying between 0 and 2 L/24 h. Two target weekly KT/V values, 1.70 and 1.90, were investigated. The equation DV/V = (5.2811-3.6996 UV)/ 17.554, derived by solving the logistic regression model, detected weekly KT/V > 1.70 (69/108, or 63.9% of the studies) with a sensitivity of 87.3% and a specificity of 60.4%. According to the same equation, the maximal UV consistent with weekly KT/V < or = 1.70 is 1.427 L/24 hr. The equation DV/V = (5.4994-2.6007 UV)/17.007 detected weekly KT/V > 1.90 (44/108, or 40.7% of the studies) with a sensitivity of 90.3% and a specificity of 79.2%. According to this equation, the maximal UV consistent with weekly KT/V < or = 1.90 is 2.115 L/24 hr. Determination of the lowest daily DV consistent with a target KT/V at any daily UV (within a wide range) is feasible in CPD.

Flow cytometric immunofluorescence assay for detection of antibodies to human immunodeficiency virus type 1 using insoluble precursor forms of recombinant polyproteins as carriers and antigens.

A new serological assay, the recombinant flow cytometric immunofluorescence assay (r-FIFA), was developed for the early detection of human immunodeficiency virus type 1 (HIV-1) antibodies by using recombinant insoluble forms of HIV-1 Gag-p45, Gag-gp41 chimeric protein, gp160, Po197 polyprotein as antigens and autologous carriers through flow cytometry. These recombinant proteins were expressed in insect cells by a baculovirus expression system. Eight anti-HIV-1 seroconversion panels, a low-titer anti-HIV-1 panel from Boston Biomedica Inc. (BBI), and three HIV-1 seroconversion specimens from the Provincial Health Laboratory of Ontario, Toronto, Ontario, Canada (PHL), were tested and analyzed by r-FIFA. In sensitivity comparisons between r-FIFA and tests licensed by the U.S. Food and Drug Administration, which were used to test all of the HIV-1 panels from BBI, detection of HIV-1 antibody by r-FIFA was on average greater than 20 days earlier than that by enzyme immunoassay. The sensitivity of r-FIFA has permitted the detection of HIV-1-specific immunoglobulin G (IgG), IgM, and IgA antibodies during seroconversion. A kinetic analysis of HIV-1 antibody production of r-FIFA has shown that either IgG or IgM, or both, can be detected, depending on the phase and type of the immune response in the HIV-1-infected individual. Both primary and secondary immune responses were observed during this period. The r-FIFA results suggest that implementation of r-FIFA may significantly reduce the "window" period from the time of infection to the time of seroconversion, with earlier detection of antibodies after initial infection. This would also make it possible for us to understand the immune response and the precise mechanisms of immunopathogenesis in the early period of HIV-1 infection.

Continuous peritoneal dialysis in heavyweight individuals: urea and creatinine clearances.

OBJECTIVE: To study whether or not continuous peritoneal dialysis (CPD) can provide acceptable levels of normalized urea and creatinine clearance in heavyweight individuals. DESIGN: Retrospective analysis of urea and creatinine clearance studies. SETTING: CPD patients followed in four dialysis units in Albuquerque, two dialysis units in Thessaloniki, and two dialysis units in Athens. PARTICIPANTS: One hundred and ninety-nine patients on CPD with 266 clearance determinations between 1991 and 1995. INTERVENTIONS: The heavyweight group consisted of 22 patients (24 clearance studies) weighing 100 kg or more (109 +/- 8.7 kg) at the time of the clearance study. All subjects were obese. The reference group consisted of 177 CPD subjects (242 clearance studies) of normal weight (68.7 +/- 12.2 kg). Urea fractional clearance (KT/V) and normalized creatinine clearance (Ccr) were compared between the heavyweight and the reference groups. MAIN OUTCOME MEASURES: The lowest acceptable weekly levels were set at 1.70 for KT/V and 54.4 L/1.73 m2 for Ccr. RESULTS: Weekly KT/V was 1.75 +/- 0.41 in the heavyweight group and 1.94 +/- 0.52 in the reference group (p = 0.047). Corresponding weekly Ccr levels were 64.0 +/- 24.3 and 77.6 +/- 40.3 L/1.73 m2, respectively (p = 0.021). In the heavyweight group, 13 studies (54.2%) had acceptable KT/V values compared to 160 studies (66.1%) in the reference group (NS). Corresponding values for acceptable Ccr were 17 (70.8%) and 165 (68.2%), respectively (NS). Drain volume was 12.96 +/- 4.40 L/24 hours in the heavyweight group and 9.63 +/- 2.58 L/24 hours in the reference group (p = 0.001). High daily exchange volume was delivered by a combination of daily continuous ambulatory peritoneal dialysis (CAPD) and nocturnal automated peritoneal dialysis (APD) in 13/16 heavyweight studies. This combination was tolerated better than any other method of delivering a large daily exchange volume. CONCLUSION: Although normalized urea and creatinine clearances are lower in obese, heavyweight individuals than in lean CPD subjects with lower weight, approximately equal percentages of these two groups achieve acceptable clearance levels. However, heavyweight individuals require larger-than-usual daily exchange volumes. The preferred way to deliver these large dialysate volumes is a combination of daily CAPD and nocturnal APD.

Fractional urea clearance estimates using two anthropometric formulas in continuous peritoneal dialysis: sex, height, and body composition differences.

OBJECTIVE: To compare estimates of urea volume (V) and KT/V obtained by the Watson and Hume anthropometric formulas, and to identify the similarities and differences between these estimates. DESIGN: Theoretical analysis applying wide variations in the determinants of anthropometric V (age, height, weight) in hypothetical women and men. Analysis of urea kinetic studies performed in patients on continuous peritoneal dialysis (CPD). SETTING: Four dialysis units in Albuquerque, two in Athens, and two in Thessaloniki. PARTICIPANTS: Three hundred and two CPD patients who had 440 urea kinetic studies. INTERVENTION: Standard urea clearance was performed by 24-hour collections of urine and drained dialysate followed by blood sampling. V was estimated by both the Watson and Hume formulas. MAIN OUTCOME MEASURES: Estimates of V and KT/V were compared separately in women and men by Student's t-test, linear regression, and limits of agreement (mean difference +/- 2 SD). The agreement of the KT/V estimates was also tested by the kappa ratio using a value of 1.70 weekly as the lowest acceptable K/TV. RESULTS: The theoretical analysis indicated important disagreement only in extreme variations from the ordinary in height and, to a lesser extent, weight. Differences due to height variation were pronounced only in hypothetical women. CPD patient findings were as follows: in women, Watson V and weekly KT/V were 30.4 +/- 4.4 L and 2.10 +/- 0.61, respectively. Corresponding Hume estimates were 30.3 +/- 5.4 Land 2.1 2 +/- 0.66, respectively. Corresponding estimates for men were 40.5 +/- 5.7 L and 1 .92 +/- 0.57 (Watson) plus 41.4 +/- 5.6 L and 1.88 +/- 0.57 (Hume), respectively. By linear regression, KT/V(Hume) = -0.083 + 1.052 (KT/V(Watson)), r = 0.961 (women); and KT/V(Hume) = -0.026 +/- 0.992 (KT/V(Watson)), r = 0.985 (men). Limits of agreement were -1.41 L and 2.10 L for V, and -0.15 and 0.14 weekly for KT/V. In 94.3% of the cases, KT/V(Watson) and KT/V(Hume) agreed (both > 1 .70 or both < 1 .70 weekly). Kappa ratio was 0.875 (excellent agreement). The concordant and discordant groups differed in height and degree of obesity, in agreement with the theoretical analysis. CONCLUSION: The Watson and Hume formulas provide similar estimates of V and KT/V in CPD patients. Differences may be noted only if women's height or, to a lesser extent, both sexes' weight is at a great variance with the ordinary values.

Fractional urea clearance in continuous ambulatory peritoneal dialysis: effects of volume disturbances.

The effects of volume disturbances on the KT/V were investigated prospectively in 133 consecutive urea clearance studies on patients on continuous ambulatory peritoneal dialysis (CAPD). Four subjects (group A) had fluid loss (actual weight < dry weight), 57 (group B) were studied on the basis of their dry weight, 58 (group C) had moderate fluid excess (0.5% of dry weight), and 14 patients (group D) had severe fluid excess (> 5% of dry weight). The KT/V was calculated on the basis of dry weight and actual weight with V obtained from the Watson anthropometric formulae which were applied either uncorrected or with a correction for changes in body water from dry weight conditions. The groups compared by variance analysis. The following weekly KT/V estimates were obtained: (1) using dry weight V: group A 1.81 +/- 0.17, group B 2.0 +/- 0.59, group C 1.79 +/- 0.46, group D 1.85 +/- 0.37 (no difference between groups); (2) using uncorrected actual weight V: group A 1.85 +/- 0.15, group B 2.05 +/- 0.59, group C 1.77 +/- 0.45, group D 1.78 +/- 0.37 (group B had a higher KT/V than group C, p < 0.05), and (3) using corrected actual weight V: group A 1.93 +/- 0.14, group B 2.05 +/- 0.59, group C 1.70 +/- 0.43, group D 1.63 +/- 0.36, (group B had a higher KT/V than either group C or group D, both at p < 0.01). In CAPD, fluid deficit is rare and causes a small increase in KT/V, whereas fluid retention is frequent and causes a decrease in KT/V. This decrease is pronounced in patients with severe fluid excess. The uncorrected Watson formulae underestimate V and, consequently, overestimate KT/V in CAPD patients with fluid excess.

Long-term continuous ambulatory peritoneal dialysis in diabetics.

Of 147 diabetic patients with end-stage renal disease who were treated in our CAPD program between 1978 and 1991, 6 men and 1 woman (5 had type II and 2 type I diabetes) with a mean age of 54 (range 21-70) years have been on CAPD for more than five years (mean: 76 mos, range: 65-109 mos) and on peritoneal dialysis (IPD+CAPD) for an average of 85 (range: 67-118) mos. They had a variety of comorbid conditions at the start of CAPD: Retinopathy (5/7), blindness (3/7), hypertension (5/7), peripheral neuropathy (7/7), peripheral vascular disease (3/7), congestive heart failure (3/7), myocardial infarction (1/7), ischemic heart disease (2/7). Two were smokers and five over the age of 65. Peritonitis rate was 1 episode/11.4 pt mos, exit-site infection 1/76.4 pt mos and average hospitalization rate 32.8 days/patient/year. Hypertension was well-controlled with discontinuation of all medications; after initiation of CAPD two of them remained without medications throughout the study but in the rest, medications had to be restarted. As assessed by HbA1c, blood glucose control improved with IP administration of insulin. Residual renal function progressively decreased. None of them developed severe hyperparathyroidism. Peripheral neuropathy remained stable in four and deteriorated in two. Total protein, albumin, cholesterol and triglycerides decreased during the last two years indicating a degree of malnutrition. Our experience with these seven patients suggests that diabetic patients, even the aged and those with many comorbid conditions and complications, can survive for long periods on CAPD.