Comparison and optimization of SAR-PAGE tests for erythropoietins in doping analysis.

Erythropoietins (EPOs) are substances listed in S2 of the World Anti-Doping Agency (WADA) Prohibited List and are used commonly by athletes to increase endurance performance. According to the current WADA Technical Documents, sarcosyl-polyacrylamide gel electrophoresis (SAR-PAGE) followed by western blotting to differentiate erythropoietins based on their molecular weights is the only method that can be used for both screening and confirmation of all types of erythropoietins. The efficiency of immunopurification and protein transfer is crucial for ensuring the selectivity and sensitivity of erythropoietin detection. Several comparisons and optimization of the SAR-PAGE tests were conducted in this study. We optimized the first blotting conditions and then compared different immunopurification methods based on their selectivity, repeatability, and sensitivity for both urine and blood analysis. Additionally, rapid procedures for both urine and blood analysis were established and compared. The two-step procedure at 1.0 mA/cm2 for 60 min followed by 1.56 mA/cm2 for 20 min increased the blotting efficiency compared with the commonly used constant current approach. Comparison of immunopurification revealed no significant difference in selectivity and sensitivity between the different methods. For other factors, such as operation complexity, time and cost, a StemCell® purification kit followed by single blotting and magnetic beads followed by double blotting are recommended for urine screening and confirmation, respectively. While magnetic beads and a MAIIA® kit followed by double blotting are recommended for both screening and confirmation of blood samples, respectively. To ensure high sensitivity and selectivity, double blotting is recommended for a rapid procedure for both urine and blood analysis.

Cost-Effectiveness Analysis of Diagnosis of Duchenne/Becker Muscular Dystrophy in Colombia.

OBJECTIVES: To determine the cost-effectiveness ratio of different courses of action for the diagnosis of Duchenne or Becker muscular dystrophy in Colombia. METHODS: The cost-effectiveness analysis was performed from the Colombian health system perspective. Decision trees were constructed, and different courses of action were compared considering the following tests: immunohistochemistry (IHC), Western blot (WB), multiplex polymerase chain reaction, multiplex ligation-dependent probe amplification (MLPA), and the complete sequencing of the dystrophin gene. The time horizon matched the duration of sample extraction and analysis. Transition probabilities were obtained from a systematic review. Costs were constructed with a type-case methodology using the consensus of experts and the valuation of resources from consulting laboratories and the 2001 Social Security Institute cost manual. Deterministic sensitivity and scenario analyses were performed with one or more unavailable alternatives. Costs were converted from Colombian pesos to US dollars using the 2014 exchange rate. RESULTS: In the base case, WB was the dominant strategy, with a cost of US $419.07 and a sensitivity of 100%. This approach remains the dominant strategy down to a 98.2% sensitivity and while costs do not exceed US $837.38. If WB was not available, IHC had the best cost-effectiveness ratio, followed by MLPA and sequencing. CONCLUSIONS: WB is a cost-effective alternative for the diagnosis of patients suspected of having Duchenne or Becker muscular dystrophy in the Colombian health system. The IHC test is rated as the second-best detection method. If these tests are not available, MLPA followed by sequencing would be the most cost-effective alternative.

Direct Immunodetection of Antigens Within the Precast Polyacrylamide Gel.

Western blotting is one of the few basic techniques widely used in the study of proteins in life science research. Despite its prevalence, the procedure has remained practically unchanged for more than 20 years. Although the method is viewed as being error-prone and as requiring excessive hands-on time, it is still widely accepted because it provides sensitive and direct information about the protein characteristics. The process is attractive to researchers because it reduces the investment in instrumentation and setup. Here we describe a procedure that eliminates the transfer step of western blotting and allows for antigen detection directly within the polyacrylamide gel, thus minimizing the investment necessary for setting up western blotting.

Application of Intermittent Microwave Irradiation to Western Blot Analysis.

We established a shortened protocol for Western blot analysis using intermittent microwave irradiation. With this method, the procedure is completed within 1 h after applying the primary antibody, and thus greatly saves time. This procedure appears to be applicable to any antibody based on our experience of several years.

A sensitive dual signal amplification method for western blotting based on antibody-functionalised graphene oxide and gold nanoparticles.

In this study, we report an ultrasensitive western blotting method using antibody-functionalised graphene oxide sheets and gold nanoparticles. Additionally, the cost is reduced greatly by conjugating two different primary antibodies on gold nanoparticles (C. Welinder and L. Ekblad, J. Proteome Res., 2011, 10, 1416-1419).

Cost effectiveness analysis of the New Jersey rapid testing algorithm for HIV testing in publicly funded testing sites.

BACKGROUND: Before 2009, New Jersey (NJ) publicly funded counseling and testing sites (CTS) tested for HIV using a single rapid test followed, when positive, by a Western Blot (WB) for confirmation. With this strategy, 74.8% of confirmed positive clients returned to receive test results. To improve the client notification rate at these centers, the New Jersey (NJ) Division of HIV, STD and TB Services (DHSTS) implemented a rapid testing algorithm (RTA) which utilizes a second, different, rapid test to verify a preliminary positive. OBJECTIVE: To compare the cost-effectiveness of the two testing algorithms. STUDY DESIGN: This was a retrospective cost-effectiveness analysis. DATA SOURCES: New Jersey HIV Rapid Testing Support Program (NJHIV) records, DHSTS grant documents, counseling time estimates from an online survey of site supervisors. Costs included test kits and personnel costs from month of RTA implementation through 11/30 in 2008 and 2009. The incremental cost of the RTA was calculated per additional percent of positive clients who were notified and per day earlier notification. RESULTS: In 2008, 215 of 247 clients with a positive rapid HIV test were confirmed positive by WB. 90.9% of clients were notified a mean of 11.4 days after their initial test. 12 refused confirmatory WB. In 2009, 152 of 170 clients with one positive rapid test had a confirmatory second positive rapid test and were notified on the same day. The incremental cost of the RTA was $20.31 per additional positive person notified and $24.31 per day earlier notification or $3.23 per additional positive person and $3.87 per day earlier notification if the WB were eliminated. CONCLUSIONS: The RTA is a cost-effective strategy achieving 100% notification of newly HIV positive clients a mean of 11.4 days earlier compared to standard testing.

An economical single-sided antibody incubation method for Western blotting.

A simple, single-sided antibody method for incubating primary and secondary antibodies in Western blotting was developed, which generates significant savings on the use of antibodies. Compared with the conventional immersion technique for antibody incubation, the present economical single-sided antibody incubation method resulted in a saving of 80% of antibody use. Besides, the present incubation method did not compromise the Western blot results and was not affected by the expression levels of target proteins.

Blue Dry Western: simple, economic, informative, and fast way of immunodetection.

The analysis by electrophoresis followed by transfer to membranes and immunodetection (Western blot) is probably the most popular technique in protein study. Accordingly, it is a time- and money-consuming procedure. Here a protocol is described where immunodetection can be accomplished in 30 min. This approach also allows permanent staining of proteins by Coomassie Blue R on the membrane before immune staining with clear background and high sensitivity.

The multiple antigen blot assay: a simple, versatile and multipurpose immunoenzymatic technique.

This technique is based on the sensitization of different antigens in a single nitrocellulose strip, which react when exposed to an immune serum and thereafter with the appropriate peroxidase conjugate and the corresponding substrate. Signals in those reactive spots are recorded as black squares in a negative photographic film, using a chemiluminiscent substrate or as blue spots when a precipitable colorimetric substrate is used. This technique allows the simultaneous demonstration of antigenicity of different antigens (peptides, recombinant molecules, and crude preparations), with a high sensitivity and specificity. Its major value is based on its versatility, since it is possible to rapidly evaluate and to compare various antigenic preparations and to use it for diagnosis of different infectious, allergic and autoimmune diseases, at a low cost.

Comparación de tres estrategias de tamizaje para la prevención de la infección perinatal por VIH en Colombia: análisis de decisiones / A comparison of three screening strategies for prevention of perinatal HIV infection in Colombia: a decision analysis model

OBJETIVO: Comparar mediante un modelo de análisis de decisiones tres estrategias de tamizaje de la infección por el VIH en mujeres embarazadas según su relación costo-efectividad y proponer la más apropiada para el sistema de salud colombiano. MÉTODOS: Estudio económico basado en el análisis mediante árboles de decisión según tres estrategias de tamizaje de la infección por el VIH en mujeres embarazadas: la voluntaria, la universal y la opcional. Se consideró a todas las mujeres colombianas embarazadas sin diagnóstico de infección por el VIH que se presentaban para el parto. Se emplearon los costos médicos directos desde la realización de la prueba hasta un año después del parto, según el Sistema General de Seguridad Social en Salud. Se compararon las razones costo-efectividad y el ahorro de cada estrategia analizada. RESULTADOS: Por cada 10 000 mujeres, la estrategia universal permitió detectar 5 casos más que la estrategia voluntaria y 7 casos más que la opcional. La estrategia universal generó costos aproximados de US$ 17,00 por cada recién nacido positivo, es decir, menos de la mitad que lo calculado para la estrategia voluntaria (US$ 38,00) y menor que para la opcional (US$ 24,00). Según el análisis bifactorial, la estrategia de tamizaje universal fue menos costosa que la voluntaria y más efectiva que las otras dos estrategias, independientemente de la prevalencia, la tasa de positivos falsos del sistema de diagnóstico empleado y la tasa de aceptación materna para realizarse la prueba de tamizaje. CONCLUSIONES: La estrategia de tamizaje voluntaria, que se utiliza actualmente en Colombia, es más costosa que la universal a mediano y largo plazos y tiene menor efectividad y capacidad de prevención. Se recomienda a las autoridades nacionales de salud realizar el tamizaje de la infección por el VIH a todas las embarazadas colombianas con pruebas de tercera generación.

OBJECTIVES: To apply decision analysis to compare the cost-effectiveness of three strategies for HIV screening of pregnant women and to recommend the one most appropriate for the health care system of Colombia. METHODS: An economic study applying decision analysis to three types of HIV screening of expectant women: voluntary, universal, and optional. All the women in Colombia with unknown HIV status who were admitted for child birth were included. The study included all the direct medical costs incurred from the time of testing through the first year following delivery, according to the General System for Healthcare Social Security. Cost-effectiveness ratio and the savings of each of the strategies were compared. RESULTS: For every 10 000 women, the universal strategy detected five cases more than the voluntary strategy and seven cases more than the optional. The universal strategy carried a cost of approximately US$ 17 for each HIV-positive newborn; that is, less than half of that of the voluntary strategy (US$ 38) and less than the optional (US$ 24). According to the bifactorial analysis, the universal screening strategy was less costly than the voluntary and more effective than both of the others, regardless of prevalence, the false-positive rate of each method, and the rate of maternal compliance with screening. CONCLUSIONS: The screening strategy currently in use in Colombia is more costly (in both the medium- and long-term), less effective, and less capable of prevention, than the universal screening strategy. The recommendation to the national health authorities of Colombia is to begin screening all pregnant women for HIV infection using third-generation testing.

[A comparison of three screening strategies for prevention of perinatal HIV infection in Colombia: a decision analysis model]. / Comparación de tres estrategias de tamizaje para la prevención de la infección perinatal por VIH en Colombia: análisis de decisiones.

OBJECTIVES: To apply decision analysis to compare the cost-effectiveness of three strategies for HIV screening of pregnant women and to recommend the one most appropriate for the health care system of Colombia. METHODS: An economic study applying decision analysis to three types of HIV screening of expectant women: voluntary, universal, and optional. All the women in Colombia with unknown HIV status who were admitted for child birth were included. The study included all the direct medical costs incurred from the time of testing through the first year following delivery, according to the General System for Healthcare Social Security. Cost-effectiveness ratio and the savings of each of the strategies were compared. RESULTS: For every 10,000 women, the universal strategy detected five cases more than the voluntary strategy and seven cases more than the optional. The universal strategy carried a cost of approximately US$ 17 for each HIV-positive newborn; that is, less than half of that of the voluntary strategy (US$ 38) and less than the optional (US$ 24). According to the bifactorial analysis, the universal screening strategy was less costly than the voluntary and more effective than both of the others, regardless of prevalence, the false-positive rate of each method, and the rate of maternal compliance with screening. CONCLUSIONS: The screening strategy currently in use in Colombia is more costly (in both the medium- and long-term), less effective, and less capable of prevention, than the universal screening strategy. The recommendation to the national health authorities of Colombia is to begin screening all pregnant women for HIV infection using third-generation testing.

Cost comparison of three HIV counseling and testing technologies.

BACKGROUND: In the United States, more than 2 million human immunodeficiency virus (HIV) antibody tests are performed annually at publicly funded HIV counseling and testing (CT) clinics. Clients do not receive results from one third of these tests because of low return rates. New rapid-testing technologies may improve receipt of results, but no study has systematically analyzed the costs of these newer technologies compared with the standard protocol. OBJECTIVE: To estimate and compare the economic costs associated with three HIV CT protocols: the standard protocol and the one-step and two-step rapid protocols. METHODS: A cost analysis model was developed in 2002 to calculate the intervention costs for HIV CT services with the standard CT protocol and the one-step and two-step rapid-test protocols for a hypothetical client in a publicly funded HIV clinic. Sensitivity analyses were performed to ascertain the effects of uncertainty in the model parameters. RESULTS: The one-step rapid protocol was generally the least expensive of the three protocols. The standard protocol cost less than the two-step protocol per HIV-positive client notified of his or her HIV status, but cost more per HIV-negative client. The sensitivity analysis indicated overlap in the cost estimates for HIV-negative clients, reflecting the generally similar costs of the three testing protocols. Taking into account HIV seroprevalence, the two-step rapid protocol would be less expensive than the standard protocol for most publicly funded testing programs in the United States. CONCLUSIONS: Rapid test protocols offer economic advantages as well as convenience, compared to the standard testing protocol. The cost estimates presented here should prove helpful to HIV program managers and other public health decision makers who need information on these counseling and testing technologies.

Estimating the costs and benefits of screening monogamous, heterosexual couples for unrecognised infection with herpes simplex virus type 2.

OBJECTIVES: Herpes simplex virus type 2 (HSV-2) is the most common cause of ulcerative genital disease in the United States, but infection is commonly unrecognised. Serological screening tests could identify discordantly infected couples and permit targeted interventions to limit HSV-2 transmission. Our objective was to evaluate the projected cost effectiveness of strategies to prevent HSV-2 transmission in couples with no history of HSV-2 infection. METHODS: We created a mathematical model to simulate the natural history and costs of HSV-2 transmission, and the expected impact of HSV-2 prevention strategies in monogamous, heterosexual couples. Strategies evaluated included (i) no screening; (ii) universal condom use; and (iii) serological screening for HSV-2 with condom use targeted to discordant couples. Screening tests considered included western blot (WB), ELISA, and ELISA with confirmation of positive test results using WB (ELISA-->WB). RESULTS: Compared to no screening, the use of ELISA-->WB prevented 38 future infections per 1000 couples, with a cost effectiveness ratio of $8200 per infection averted. The use of WB in all couples had an incremental cost effectiveness ratio of $63 600 per infection averted. Strategies of ELISA alone and universal condom use were not cost effective. The cost effectiveness of ELISA-->WB improved with increasing prevalence of HSV-2, but worsened with decreasing condom compliance. Screening with ELISA alone was a reasonable strategy only when ELISA specificity increased to 99%. CONCLUSIONS: Serological screening for unrecognised HSV-2 infection in monogamous, heterosexual couples is expected to decrease the incidence of HSV-2 infection, but increase healthcare costs. For couples choosing to be screened, a two step testing strategy (ELISA-->WB) is recommended. Recommendations for a national policy to conduct serological screening will depend on the value placed on averting an incident HSV-2 infection.

The impact of expected HIV transmission rates on the effectiveness and cost of ruling out HIV infection in infants.

OBJECTIVE: To quantify the costs and effectiveness of different strategies for ruling out HIV infection in infants born to HIV-infected mothers in the United States. METHODS: The authors assessed 4 different testing strategies that incorporated serial HIV DNA polymerase chain reaction (PCR) testing with or without enzyme-linked immunosorbent assay (ELISA) antibody testing. Testing costs, false reassurance rates, and incremental cost-effectiveness ratios were compared for the 4 strategies. RESULTS: In HIV-exposed infants, HIV DNA PCR testing at birth, 1 month, and 4 months of age results in a false reassurance rate of 21 per million (at a 2% transmission rate). Adding an ELISA test lowers the false reassurance rate to 0.052 per million at a cost of $570,000 per additional case detected; adding another PCR lowers the false reassurance rate to 1.49 per million at a cost of $720,000 per additional case detected compared with the 3-PCR strategy. At a high transmission rate (20%), there would be substantially more erroneously negative results (false reassurance rate is 256 per million with PCR testing at birth, 1 month, and 4 months) and consequently more favorable cost-effectiveness ratios with additional testing: $47,000 per additional case detected by adding 1 ELISA test and $59,000 per additional case detected by adding another PCR test. CONCLUSIONS: False-negative HIV results after serial testing in exposed infants are rare, and the incremental cost-effectiveness ratios of additional tests are substantial at low transmission rates. However, the false reassurance rate increases considerably with a 3-PCR strategy and additional testing becomes more cost-effective at greater transmission rates; therefore, additional testing may be warranted in infants at greater risk of infection.

Strategies for diagnosis of HTLV-I and -II.

BACKGROUND: No gold standard exists for diagnosis of HTLV infection. The aim of thus study was to compare the accuracy of a combination of two sensitive ELISAs with Western blot (WB), a line immunoassay, and PCR for diagnosis of HTLV infection. STUDY DESIGN AND METHODS: Nine hundred eighty-five specimens were tested for the presence of HTLV antibodies by HTLV-I and/or HTLV-II EIAs (Murex and Ortho), WB (Diagnostic Biotechnology), line immunoassay (INNO-LIA, Innogenetics), and/or presence of HTLV DNA by PCR. The results were compared with the probable HTLV infection status of each subject, as determined by detailed review of all available laboratory, clinical, and epidemiologic data. RESULTS: The sensitivity for diagnosis of HTLV-I infection was high for all assays evaluated, but both PCR and WB had a lower sensitivity rate (approx., 80%) for confirmation of HTLV-II. INNO-LIA detected 94 percent of the HTLV-II-positive samples. However, Murex EIA in combination with Ortho EIA was 100-percent sensitive for the detection of both HTLV-I and HTLV-II antibodies. Furthermore, the number of samples giving indeterminate results in the ELISA combination was much lower as compared with WB (2.5% vs. 50%). CONCLUSION: Based on these findings, a new, more sensitive and specific test strategy for HTLV diagnosis than the current algorithm, which includes WB, is proposed. Thereby, both the direct and indirect costs can be substantially reduced.

Rational testing of the HIV-exposed infant.

OBJECTIVES: The objectives of this study were 1) to evaluate testing regimens of human immunodeficiency virus (HIV)-exposed infants and 2) to determine optimal methods of follow-up by enzyme-linked immunosorbent assay (ELISA) testing. METHODS: We reviewed the results from 742 HIV-exposed infants in the state of North Carolina; 2474 samples were tested for HIV by DNA polymerase chain reaction (PCR) at the University of North Carolina Retrovirology Core Laboratory. We then reviewed the utility and costs of ELISA testing of all HIV-exposed infants who were seen at the Duke University Pediatric Infectious Disease Clinic between January 1, 1993, and May 5, 1998. We used likelihood ratios to model probability of HIV infection given 3 negative DNA (PCR) tests and to provide recommendations on the use of ELISA follow-up. RESULTS: The overall sensitivity of the DNA PCR was 87.1%, and its specificity was 99.9%. We evaluated 224 HIV-exposed infants who were seen at Duke University and who had at least 3 negative diagnostic tests using either DNA PCR tests or HIV blood cultures. All 178 infants who subsequently underwent ELISA testing ultimately demonstrated seroreversion. The Duke University Pediatric Infectious Disease Clinic transferred the care of 65 patients to primary care physicians before ELISA testing and retained the care of the remaining 159 patients. Children who remained in Duke's care were more likely to have documentation of seroreversion (158 of 159 vs 20 of 65). We reviewed costs of travel, physician appointment, and HIV antibody testing in a tertiary care setting. Given 3 negative PCR tests, the expected cost per case of HIV detected by a positive ELISA assay is $23.8 million. CONCLUSIONS: Documentation of seroreversion in this cohort was nearly complete in the multidisciplinary subspecialty clinic but not when such responsibility was left to the primary care physician. Given the low probability of disease in patients who have had 3 negative PCR tests, documentation of a negative ELISA may not be an appropriate use of medical resources.

Screening for human T cell leukaemia/lymphoma virus among blood donors in Sweden: cost effectiveness analysis.

OBJECTIVE: To analyse the cost effectiveness of a national programme to screen blood donors for infection with the human T cell leukaemia/lymphoma virus. DESIGN: Three models for calculating the costs and benefits of screening were developed. The first model analysed the cost of continuously testing all donations; the second analysed the cost of initially testing new blood donors and then retesting them after five years; the third analysed the cost of testing donors only at the time of their first donation. Patients who had received blood components from donors confirmed to be infected with the virus were offered testing. SETTING: Sweden. MAIN OUTCOME MEASURES: Prevalence of infection with the virus among blood donors, the risk of transmission of the virus, screening costs, and the outcome of infection. RESULTS: 648 497 donations were tested for the virus; 1625 samples tested positive by enzyme linked immunosorbent assay. 6 were confirmed positive by western blotting. The prevalence of infection with the virus was 2/100 000 donors. 35 patients who had received blood infected with the virus were tested; 3 were positive. The cost of testing every donation was calculated to be $3.02m (1.88m pounds); this is 18 times higher than the cost of testing new donors only, and only 1 additional positive donor would be discovered in 7 years. Regardless of the model used, screening was estimated to prevent only 1 death every 200 years at a minimum cost of $36m (22.5m pounds). CONCLUSION: Based on these estimates the Swedish National Board of Health and Welfare decided that only new blood donors would be screened for infection with the virus.

Laboratory evaluation in the diagnosis of Lyme disease.

PURPOSE: To provide a qualitative evaluation of the predictive value of the laboratory diagnosis of Lyme disease and to use the resultant data to formulate guidelines for clinical diagnosis. DATA SOURCES: A MEDLINE search of English-language articles or articles with English-language abstracts published from 1982 to 1996. DATA EXTRACTION: Sensitivity, specificity, and likelihood ratios were calculated, and a random-effects model was used to combine the proportions from the eligible studies. Prespecified criteria were used to determine which studies were eligible for analysis. DATA SYNTHESIS: Laboratory testing in general is not clinically useful if the pretest probability of Lyme disease is less than 0.20 or greater than 0.80. When the pretest probability is 0.20 to 0.80, sequential testing with enzyme-linked immunosorbent assay and Western blot is the most accurate method for ruling in or ruling out the possibility of Lyme disease. CONCLUSIONS: Laboratory testing is recommended only in patients whose pretest probability of Lyme disease is 0.20 to 0.80. If the pretest probability is less than 0.20, testing will result in more false-positive results than true-positive results; a negative test result in this situation effectively rules out the disease.

Utilization and cost of serologic tests for Lyme disease in Maryland.

To ascertain use of serologic tests for Lyme disease (LD) in Maryland, all laboratories registered with the State Health Department were surveyed. Results show that from 1992 to 1995, 17 laboratories performed 100,000 serologic tests costing $7.1 million on Maryland residents; 90% of these tests were EIAs. The proportion of positive EIAs increased from 3.4% in 1992 to approximately 7.0% in 1994 and 1995, and the percentage of positive second tests (Western blot, WB) fell from 7.9% to 5.0%-5.5%. The large number of EIAs performed in comparison with the low incidence of LD in the state results in a low predictive value of a positive EIA test. Therefore, the WB is indicated to confirm equivocal and positive EIA tests when characteristic clinical findings of LD are not present. The 30,000 tests for LD performed annually on Maryland residents at a cost of over $2 million in direct medical costs must be added to the public health burden of LD in this state.