Dengue fever. An Australian perspective.

BACKGROUND: An estimated 2.5 billion people live in areas where dengue fever is endemic with around 100 million cases per year. A more serious form of the disease called dengue haemorrhagic fever (DHF) is the most common cause of hospitalisation and death among children in some Southeast Asian countries. OBJECTIVE: This article reviews the ecology and the pathogenesis of the disease and its clinical features. The history of dengue fever in Australia will be presented and the prospects for the future discussed. DISCUSSION: Dengue fever was common in Australia in the late 19th century and the first clinical description of DHF were published by an Australian clinician in Charters Towers north Queensland. Since that time there have been sporadic outbreaks of dengue fever. In recent years there has been an increase in the number of epidemics in north Queensland. If Australia follows a pattern observed elsewhere in the world there might again be cases of DHF recorded on Australian soil.

Conversion of cardiac and liver transplant recipients from HPLC and FPIA (polyclonal) to an FPIA (monoclonal) technique for measurement of blood cyclosporin A.

In an effort to replace HPLC and FPIA (polyclonal) for whole blood determination of Cyclosporin A (CsA), this study examined the application of FPIA (monoclonal) in patients post cardiac and liver transplantation. The assay had a minimum detectable dose of 15 microg/L, an overall recovery of 97% and was linear to 1200 microg/L, and gave inter-assay precision values of < 5% (CV). On comparing FPIA (monoclonal) and HPLC for 59 cardiac transplant recipient blood samples, a correlation of FPIA (monoclonal) = 1.30 (HPLC) + 36.34, r = 0.96 was obtained. With liver transplant samples (n = 348), the correlation was FPIA (monoclonal) = 1.21 (HPLC) + 42.15, r = 0.98. Correlation on 131 cardiac transplant recipients gave FPIA (monoclonal) = 0.31 FPIA (polyclonal) + 43.97, r = 0.68. It is concluded that when converting from HPLC to FPIA (monoclonal) a positive bias of 21%-30% is observed, and in replacing FPIA (polyclonal) with FPIA (monoclonal), a negative bias of 50%-69% is seen with liver and cardiac patients respectively. These data indicate that therapeutic ranges should be re-established or adjustments in CsA dosing would be necessary.

Immunochemical extraction and automated measurement of plasma creatine kinase MB isoenzyme and creatine kinase MB2 isoform.

Measurement of creatine kinase MB (CK-MB) and its isoforms CK-MB2 and CK-MB1 are now applied in the diagnosis of acute myocardial infarction (AMI). The most common approach for analysis includes RIA, IRMA, and electrophoresis, all of which may be time-consuming. This study examines determination of CK-MB and CK-MB2 by a rapid immunochemical extraction method followed by an automated measurement for both analytes. The automated method was sensitive to 2 U/L, linear to 180 U/L, and gave excellent interassay precision (< 10% CV). Interference studies indicated that bilirubin, hemolysis, and lipemia caused analytical problems as did the presence of high activities of other CK isoenzymes, notably CK-MM and CK-BB, requiring dilution of samples prior to analysis. Application of immunochemical extraction gave a reference interval of CK-MB (0-2.5 U/L) and CK-MB2 (0.1-1.4 U/L) for blood donors (20-60 years), peak levels for ruled-out AMI patients of CK-MB (0.5-7.3 U/L) and CK-MB2 (0.3-4.9), peak levels for ruled-in AMI patients of CK-MB (80-174 U/L) and CK-MB2 (80-155 U/L). Coronary artery bypass patients (n = 24) and all trauma patients (n = 14) also demonstrated elevations in CK-MB and CK-MB2, whereas only five of the trauma patients demonstrated increased CK-MB by IRMA. In patients (n = 7) having increased total CK and normal CK-MB by IRMA, the extraction assay for CK-MB and CK-MB2 yielded increased values in all patients. This new approach to CK-MB and CK-MB2 analysis can be performed within 30 minutes of sample receipt.

Suramin in hormone-refractory metastatic prostate cancer: a drug with limited efficacy.

PURPOSE: To confirm the previously reported high response rates and prolonged survival in hormone-refractory prostate cancer treated with suramin. PATIENTS AND METHODS: Thirty-six eligible patients with hormone-refractory prostate cancer with either measurable disease or bone disease only and a prostate-specific antigen (PSA) level greater than 50 ng/mL were enrolled. Treatment consisted of two 8-week courses of outpatient-based therapy with an interposed rest period. A bayesian adaptive control strategy and a three-compartment pharmacokinetic model that accommodates clearance changes was used to guide individual dosing. A rapid infusion of 1,000 mg/m2 suramin was followed by five daily infusions that targeted 285 micrograms/mL peak plasma levels during the first week. All patients received concomitant hydrocortisone. For the next 7 weeks, patients received one to two doses per week that targeted levels in the 150 to 285 micrograms/mL range and integrated weekly averages of 200 ug/mL. RESULTS: Nine patients (28%) had a partial response to suramin based on a > or = 50% decrease in PSA levels coupled with either relief of bone pain or by a 50% decrease in measurable disease. The median overall survival time for all patients is 31 weeks (95% confidence interval [CI], 23 to 51). Treatment was generally well tolerated, with fatigue being the most common significant toxicity, but fatal idiosyncratic myelosuppression (grade V) was observed in one patient. CONCLUSION: Using this dosing schedule, suramin has limited activity against hormone-refractory metastatic prostate cancer. Recent data suggest that hydrocortisone administered with suramin may be partly responsible for the benefit attributed to the drug. Although a small cohort of patients appeared to benefit, we were unable to confirm the previously reported high rate of activity and durability of remission using this agent.

Analysis of whole-blood cyclosporin G by liquid chromatography in renal transplant recipients.

Cyclosporin G (CsG) is less nephrotoxic than Cyclosporin A (CsA) and is undergoing clinical trials for use as an immunosuppressive agent after renal transplantation. In this study, CsG was measured by a rapid high-performance liquid chromatography (HPLC) technique in blood samples (n = 107) received from renal transplant recipients. The HPLC assay proved to be analytically suitable in that it was sensitive, linear, and precise and had high recovery (102%). However, interference was observed from some potentially co-administered drugs such as calcitriol, ferrous sulfate, hydrazaline, and minoxidil. The HPLC assay for CsG correlated well with a FPIA (Abbott TDx), FPIA = 0.964 (HPLC) + 33.59, r = 0.9819, Sy/x = 36.66 for patients receiving a low dose of CsG (5 mg/kg/day) and a high dose (10 mg/kg/day). Furthermore, the HPLC technique was capable of measuring predictable CsG concentrations when the drug was tapered to lower doses at various stages of the 16 week clinical trial. The HPLC for CsG has the further advantage that the same system and mobile phase can be used to measure CsA while using CsC as the interval standard.

Evaluation of reference ranges for fatty acids in serum.

Long chain fatty acid fractionation has become a valuable tool in the management of patients maintained on total parenteral nutrition. While many clinicians prefer to use absolute concentrations to monitor a patient's fatty acid status, reference ranges are not available. Previous reference range studies reported values in terms of percentages only and calculated ranges parametrically. However, due to the non-Gaussian distributions of some serum fatty acids, it is necessary to calculate reference ranges non-parametrically. Serum from blood donors (n = 130) were collected and analyzed for total fatty acids by gas-liquid chromatography. Results for each of the fatty acids were calculated both as a concentration and as a percentage of the total fatty acids measured.

Whole-blood cyclosporin G in renal transplant recipients determined by two immunoassays and liquid chromatography.

Cyclosporin G (CsG) is less nephrotoxic than cyclosporin A (CsA) and is undergoing clinical trials for use as an immunosuppressive agent after renal transplantation. Three assays for whole-blood CsA-HPLC, RIA (INCSTAR, Cyclo-Trac SP), and FPIA (Abbott TDx)--were adapted for use with CsG and were assessed for analytical suitability and to determine which assay was capable of deriving CsG values rapidly after transplantation. The assays were acceptable in terms of sensitivity, linearity, analytical recovery, and precision. When considering blood samples (n = 107) from renal transplant recipients receiving a low dose of CsG (5 mg/kg per day) and a high dose (10 mg/kg per day), we obtained the following correlation data: RIA = 0.974HPLC + 27.89 (r = 0.9798, Sy/x = 39.24); FPIA = 0.964HPLC + 33.59 (r = 0.9819, Sy/x = 36.66); and FPIA = 0.977RIA + 9.50 (r = 0.9894, Sy/x = 28.12). The FPIA of CsG is recommended as the most rapid method, although it is the most expensive. HPLC, RIA, and FPIA were capable of accurately deriving projected CsG concentrations at various stages of the clinical trial when the low- and high-dose regimes were tapered over a period of 16 weeks.

Measurement of cyclosporine by liquid chromatography and three immunoassays in blood from liver, cardiac, and renal transplant recipients.

In an effort to replace HPLC for whole-blood determination of cyclosporine (CsA), we compared HPLC with radioimmunoassay (RIA; INCSTAR, Cyclo-Trac SP assay), fluorescence polarization immunoassay (FPIA; Abbott TDx), and in-house modified enzyme-multiplied immunoassay technique (EMIT; Syva Co.). For blood samples from 200 various transplant recipients, RIA = 1.262 (HPLC) - 8.16, r = 0.983; FPIA = 1.200 (HPLC) + 19.90, r = 0.981; and EMIT = 1.038 (HPLC) + 11.28, r = 0.985. For segregation by transplant type, RIA, FPIA, and EMIT demonstrated positive biases of 27%, 12%, and 3%, respectively, for liver transplant recipients (n = 50) when compared with HPLC. Heart transplant recipients (n = 50) gave positive bias values of 23%, 14%, and 4% for RIA, FPIA, and EMIT, respectively. Adult renal transplant recipients (n = 50) demonstrated positive bias values of 30%, 31%, and 0% for RIA, FPIA, and EMIT, respectively. For pediatric renal transplant recipients (n = 50), positive biases of 40%, 31%, and 9% were obtained for RIA, FPIA, and EMIT, respectively. We conclude that the modified EMIT represents the best replacement for HPLC.

Prevalence of non-A,non-B hepatitis/hepatitis C virus antibody in human immunoglobulins.

Human intravenous immunoglobulins prepared by the cold ethanol fractionation technique of Cohn are considered safe with respect to infectivity. However, there have been several instances of transmission of both hepatitis B and non-A,non-B hepatitis viruses after administration of intravenous immunoglobulins. To determine the prevalence of hepatitis C virus antibody in intravenous immunoglobulins and protein preparations, 30 commercially available products were tested. Using the Abbott enzyme immunoassay for hepatitis C virus antibody, 27 of 30 (90%) immunoglobulins tested positive. The Ortho immunoassay showed that 28 of 30 (93%) were positive, with one discordant result between the Ortho and Abbott assays. An antigen-blocking or neutralization test (Abbott) confirmed the results of the Ortho assay. Bovine, sheep, goat, and horse sera also were tested before and after isolation of animal immunoglobulins. All results on the animal sera were negative, indicating that the fractionation process did not produce false-positive results. The high prevalence rate of hepatitis C virus antibody in intravenous immunoglobulins has important implications for follow-up of recipients, selection of serum donors, and implementation of anti-hepatitis C virus testing.

Comparison of various methods for the enumeration of blood cells in urine.

This study compared the use of conventional urine microscopy, the KOVA system, Neubauer hemacytometry, and the Yellow IRIS for the determination of white blood cell (WBC) and red blood cell (RBC) counts in urine samples. Both KOVA WBC and RBC counts correlated better with the IRIS counts than with conventional microscopy. KOVA WBC counts correlated with Neubauer hemacytometry to the same degree as they did with IRIS WBC counts. The RBC count correlation was fairly similar between KOVA, hemacytometry, and the conventional method. It was concluded that the KOVA system is a suitable replacement for conventional urine microscopy.

Creatine kinase MB measured by fluorometric enzyme immunoassay and immunochemiluminescence.

Creatine kinase MB (CK-MB) was measured in serum by a fluorometric enzyme immunoassay on the Stratus analyzer and by an immunochemiluminometric assay using the Ciba Corning Magic Lite System. Both methods were standardized against purified CK-MB, with Stratus underestimating by 20 percent and Magic Lite overestimating by 28 percent. The assays proved sensitive and linear; however, at a CK-MB concentration of 7.0 micrograms per L, Stratus gave unacceptable inter-assay precision. No cross-reactivity was observed with CK-MM or CK-BB and elevated triglycerides, bilirubin, and hemoglobin did not interfere. Correlations with an immunoradiometric assay (Embria), using 522 samples, gave: Stratus = 0.999 (Embria) -3.3; r = 0.969, and Magic Lite = 1.225 (Embria) -3.03; r = 0.971. When using Magic Lite, results from 40 acute myocardial infarction (AMI) patients gave a mean CK-MB value of 93.8 micrograms per L (range: 9.2 to 428 micrograms per L) at the peak of enzyme release and a mean value of 69.6 micrograms per L (range: 6.7 to 319 micrograms per L) when using Stratus. Both methods proved to be highly sensitive and specific in the diagnosis of AMI; however, the need for standardization of CK-MB assays is stressed.

Prevalence of non-A, non-B hepatitis/hepatitis C virus antibody in laboratory quality-assurance sera.

Quality-assurance sera (QAS) are prepared from pooled sera composed of thousands of individual donations. Previous studies documented that a substantial percentage of individual QAS test positive for viral disease markers, including antibodies to human immunodeficiency virus and to hepatitis B surface antigen. We tested 239 QAS from various proficiency programs and commercial sources to determine the prevalence of hepatitis C virus (HCV) antibody. We tested samples for anti-HCV by using an enzyme immunoassay (EIA; Abbott Labs.) and an enzyme-linked immunosorbent assay (ELISA; Ortho Diagnostics). We observed an overall positive rate of 49% by one or both assays in all categories of sera tested. In addition, we found a greater rate of positivity (58%) in proficiency program samples than in commercial samples (43%). We found discrepant results between the two assays for 15 of 239 samples (6%). In the discrepant samples, the EIA result was positive, whereas the ELISA result was negative. Anti-HCV positivity in QAS has important implications for laboratory personnel handling these samples.

Rapid liquid-chromatographic method for simultaneous determination of plasma prednisone, prednisolone, and cortisol in pediatric renal-transplant recipients.

A rapid liquid-chromatographic method is described for simultaneously determining prednisone, prednisolone, and cortisol in plasma. Before chromatography, samples containing an internal standard (methylprednisolone) were extracted with use of Chem Elut (Analytichem) columns. After elution of the glucocorticosteroids, the eluates were dried and reconstituted in a mobile phase of tetrahydrofuran/water (25/75 by vol). Samples were injected onto a reversed-phase C18 column, and analysis time was 15.5 min. Measures of analytical performance were all acceptable, and the method was used to assess noncompliance in pediatric renal-transplant recipients who were receiving prednisone and cyclosporine. Of the 37 pediatric patients tested, five (13.5%) were identified as noncompliant. The method is simple, accurate, and precise, and other steroids and medications commonly given to transplant recipients do not interfere with it.

Assessment of biological reagents for presence of human immunodeficiency virus-1 (HIV-1) antigens.

We tested 122 biological reagents including laboratory quality assurance sera and therapeutic human immune serum globulin products using an immunoenzymometric assay (IEMA) for HIV-1 antigens. Biological reagents tested were 64 HIV-1 antibody non-reactive and 44 HIV-1 antibody reactive quality assurance samples, and 14 HIV-1 antibody reactive human immune serum globulins from 21 manufacturers. Twenty-one of these biological reagents were previously reported by us as Western blot reactive. All 122 samples tested were non-reactive for HIV-1 antigens. Low incidence of HIV-1 antigen in these biological reagents should not alter laboratory safety practices in which all samples are considered infectious. Use of HIV-1 antigen measurements, either alone or with HIV-1 antibody determinations does not increase the likelihood of detecting HIV-1 reactive samples.

Human, rabbit, bovine, and porcine creatine kinase isoenzymes are glycoproteins.

Human, rabbit, bovine, and porcine creatine kinase (CK) isoenzyme preparations were extensively purified by isoelectric focusing and high-performance liquid chromatography and tested for the presence of carbohydrate. In this study, all the CK isoenzymes demonstrated positive periodic acid-Schiff (PAS) reactions, indicating the presence of carbohydrate. It is concluded that CK is a glycoprotein as a carbohydrate, associated with the M subunit of human, rabbit, bovine, and porcine isoenzymes. Also, carbohydrate is an integral part of the B subunit of both rabbit brain and porcine heart CK. Loss of carbohydrate may be important in enzyme catabolism, yielding a pool of circulating modified CK protein that, to date, has remained undetected by traditional methods.

Clinical assessment of the Hitachi 736-30 chemistry analyzer.

We assessed the Hitachi 736-30 as a possible replacement for the SMAC I and as a laboratory cost-saving measure. For 24 analytes, both intra- and interassay precisions were acceptable; they also had good measuring ranges. Essentially no interference from lipemia was observed, while minimal interference from bilirubin was demonstrated. Hemoglobin interfered in the measurement of 12 of the analytes. Correlation with the SMAC I, Demand, Astra-8, ACA, and Varian Atomic Absorption Spectrophotometer was found to be acceptable, except for chloride which showed poor correlation with SMAC I and Astra-8 (Hitachi = 0.888 [SMAC] + 11.102, r = 0.9652; Hitachi = 0.885 [Astra] + 10.264, r = 0.9136). The Hitachi 736-30 offers reagent and method flexibility, high volume capability, and "walk-away" operation.

Measurement of cyclosporine in plasma from patients with various transplants: HPLC radioimmunoassay with a specific monoclonal antibody compared.

This study compares cyclosporin A (CsA) concentrations in plasma from patients receiving various transplants, as measured by HPLC and RIA with a monoclonal antibody for CsA and an 125I-labeled ligand. The RIA was restandardized with in-house standards because it overestimated CsA by an average of 23%. The RIA was sensitive to 2 micrograms/L, the standard curve was linear from 20 to 500 micrograms of CsA per liter, analytical recovery was 98%, and CVs were less than 8% for intra- and interassay precision. RIA (y) vs HPLC (x) for 283 plasma samples from 145 patients gave a slope = 1.1256, r = 0.979. When the results were segregated according to transplant type, CsA in liver and heart recipients was overestimated by RIA as compared with HPLC: slope = 1.202, r = 0.973 and slope = 1.1477, r = 0.983, respectively. Adult and pediatric CsA values were acceptable when RIA and HPLC were compared: slope = 1.0755, r = 0.977 and slope = 1.0563, r = 0.980, respectively. For six samples (four heart, two liver recipients) where HPLC and RIA values demonstrated wide discrepancies, repeat HPLC and analysis of eluate fractions gave CsA concentrations nearer values by the initial HPLC assay. We conclude that this RIA cannot be substituted for HPLC in the case of heart and liver recipients. The need for each laboratory to standardize the RIA is obvious.

Standardization for four protein analytes with the Behring Nephelometer.

Owing to the generally higher values observed in the initial establishment of immunoglobulins (Ig) G, A, and M assays with the Behring Nephelometer, we elected to verify five commercial protein calibrators. This initial verification was performed by standardizing the Behring Nephelometer with the World Health Organization (WHO) International Reference Preparation for Human Serum Immunoglobulins G, A, and M. The instrument was also standardized for immunoglobulins and transferrin with use of the Reference Preparation for Serum Proteins (RPSP II). Analytical recoveries of the commercial calibrators varied. Also, assigned protein concentrations in both the WHO and RPSP II preparations made them unacceptable to us as benchmark calibrators for the Behring Nephelometer. Individual proteins (IgG, IgA, IgM, and transferrin) were obtained and primary standards prepared. Both transferrin and IgG were successfully standardized. However, IgA and IgM primary standards lacked 100% antigenicity, requiring removal of nonreactive IgA and IgM or reassignment of the correct value. The problem remains to find appropriate purified materials for IgA and IgM standardization.