External financial aid to blood transfusion services in sub-Saharan Africa: a need for reflection.

Jean-Pierre Allain and colleagues argue that, while unintended, the foreign aid provided for blood transfusion services in sub-Saharan Africa has resulted in serious negative outcomes, which requires reflection and rethinking.

Assessment of the heat stability of seven rapid HIV assays.

Human Immunodeficiency Virus Rapid Diagnostic Tests (HIV RDTs) are robust, quick to perform, effective diagnostic tools. The stability of seven commonly used RDTs for detecting antibody to HIV was assessed during exposure to temperatures above 30°C, the usual maximum recommended by manufacturers. The aim of the study was to determine if HIV RDTs retain their testing outcomes after exposure to higher temperatures. At two testing sites, seven RDTs were exposed to a short heat shock (60°C for 72 hours) as might occur during transport. RDTs were exposed to ambient (22 or 30°C), warm (35 or 37°C) or hot (45°C) temperatures for up to 90 days. Testing was performed at five intervals on a panel of seven positive and three negative plasma samples. Results showed no changes consistent with altered testing outcomes over time and/or temperature when test indicators were compared with the control indicators. Only one HIV RDT achieved 100% consensus with reference results at all four storage temperatures (median 97.5%, lowest 74% for RDT5 at 45°C). Testing outcomes in a limited sample panel showed six of seven HIV RDT kits were relatively robust despite exposure to higher than recommended temperatures.

Photographed rapid HIV test results pilot novel quality assessment and training schemes.

HIV rapid diagnostic tests (RDTs) are now used widely in non-laboratory settings by non-laboratory-trained operators. Quality assurance programmes are essential in ensuring the quality of HIV RDT outcomes. However, there is no cost-effective means of supplying the many operators of RDTs with suitable quality assurance schemes. Therefore, it was examined whether photograph-based RDT results could be used and correctly interpreted in the non-laboratory setting. Further it was investigated if a single training session improved the interpretation skills of RDT operators. The photographs were interpreted, a 10-minute tutorial given and then a second interpretation session was held. It was established that the results could be read with accuracy. The participants (n=75) with a range of skills interpreted results (>80% concordance with reference results) from a panel of 10 samples (three negative and seven positive) using four RDTs. Differences in accuracy of interpretation before and after the tutorial were marked in some cases. Training was more effective for improving the accurate interpretation of more complex results, e.g. results with faint test lines or for multiple test lines, and especially for improving interpretation skills of inexperienced participants. It was demonstrated that interpretation of RDTs was improved using photographed results allied to a 10-minute training session. It is anticipated that this method could be used for training but also for quality assessment of RDT operators without access to conventional quality assurance or training schemes requiring wet samples.

High viral fitness during acute HIV-1 infection.

Several clinical studies have shown that, relative to disease progression, HIV-1 isolates that are less fit are also less pathogenic. The aim of the present study was to investigate the relationship between viral fitness and control of viral load (VL) in acute and early HIV-1 infection. Samples were obtained from subjects participating in two clinical studies. In the PULSE study, antiretroviral therapy (ART) was initiated before, or no later than six months following seroconversion. Subjects then underwent multiple structured treatment interruptions (STIs). The PHAEDRA study enrolled and monitored a cohort of individuals with documented evidence of primary infection. The subset chosen were individuals identified no later than 12 months following seroconversion to HIV-1, who were not receiving ART. The relative fitness of primary isolates obtained from study participants was investigated ex vivo. Viral DNA production was quantified using a novel real time PCR assay. Following intermittent ART, the fitness of isolates obtained from 5 of 6 PULSE subjects decreased over time. In contrast, in the absence of ART the fitness of paired isolates obtained from 7 of 9 PHAEDRA subjects increased over time. However, viral fitness did not correlate with plasma VL. Most unexpected was the high relative fitness of isolates obtained at Baseline from PULSE subjects, before initiating ART. It is widely thought that the fitness of strains present during the acute phase is low relative to strains present during chronic HIV-1 infection, due to the bottleneck imposed upon transmission. The results of this study provide evidence that the relative fitness of strains present during acute HIV-1 infection may be higher than previously thought. Furthermore, that viral fitness may represent an important clinical parameter to be considered when deciding whether to initiate ART during early HIV-1 infection.

Immunoassays for the diagnosis of HIV: meeting future needs by enhancing the quality of testing.

Immunoassays for detecting HIV infection perform better than other serological assays. HIV immunoassays are presented in a number of different formats: instrument-based, plate, rapid assays and as immunoblots. HIV immunoassays for screening and diagnosis are now in their fourth generation; an assay generation meaning that significant modifications to the assay format have led to a significant enhancement in quality. Although still not perfect, they are now of exceptionally high quality if conducted properly. Most problems relate to how the assays are performed. Many laboratories, especially in high human development index (HDI) countries, manage testing within functioning quality-management systems, but this is not true of laboratories in low HDI countries or in many medium HDI countries. Simple rapid tests for HIV are being used increasingly, and create special challenges for assuring quality. Users of HIV immunoassays are learning that a poorer assay used well has better outcomes than a splendid assay performed poorly. Experience highlights the importance of conducting HIV testing within quality-managed systems and according to international standards, but testing quality and laboratory quality management must be funded adequately.

TREAT Asia Quality Assessment Scheme (TAQAS) to standardize the outcome of HIV genotypic resistance testing in a group of Asian laboratories.

The TREAT Asia (Therapeutics, Research, Education, and AIDS Training in Asia) Network is building capacity for Human Immunodeficiency Virus Type-1 (HIV-1) drug resistance testing in the region. The objective of the TREAT Asia Quality Assessment Scheme - designated TAQAS - is to standardize HIV-1 genotypic resistance testing (HIV genotyping) among laboratories to permit rigorous comparison of results from different clinics and testing centres. TAQAS has evaluated three panels of HIV-1-positive plasma from clinical material or low-passage, culture supernatant for up to 10 Asian laboratories. Laboratory participants used their standard protocols to perform HIV genotyping. Assessment was in comparison to a target genotype derived from all participants and the reference laboratory's result. Agreement between most participants at the edited nucleotide sequence level was high (>98%). Most participants performed to the reference laboratory standard in detection of drug resistance mutations (DRMs). However, there was variation in the detection of nucleotide mixtures (0-83%) and a significant correlation with the detection of DRMs (p<0.01). Interpretation of antiretroviral resistance showed approximately 70% agreement among participants when different interpretation systems were used but >90% agreement with a common interpretation system, within the Stanford University Drug Resistance Database. Using the principles of external quality assessment and a reference laboratory, TAQAS has demonstrated high quality HIV genotyping results from Asian laboratories.

Transfusion medicine and safety.

Advances in safety of blood transfusion in clinical practice principally relate to preventing transfusion-transmitted infections (TTI). Epidemiological studies of TTI have resulted in the development, standardization, and implementation of an expanding array of immunoassays employed worldwide in routine screening of blood donated by voluntary blood donors. Exclusion of infected blood and their donors has remarkably reduced the risk of transmitting HBV, HCV, HIV-1/2, and HTLV-I/II infections. Nucleic acid tests (NAT) using enzymatic amplification of viral gene sequences have augmented the risk reduction in "window period" infections that are undetectable by the serological tests. Improved viral safety of transfusion therapy has led us to recognize the risk of bacterial contamination, especially in platelet concentrates stored optimally at room temperature. Besides the current effort devoted to microbial risk reduction, pathogen inactivation technologies promise reduction of the residual risk of known and emerging infectious agents. The clinical effectiveness of the foregoing measures, international harmonization/standardization of practices and procedures, and continued hemovigilance portend safest possible safety in the clinical practice of blood transfusion.

Evaluation of eight anti-rubella virus immunoglobulin g immunoassays that report results in international units per milliliter.

An evaluation of anti-rubella virus immunoglobulin G (IgG) immunoassays that report in international units per milliliter (IU/ml) was performed to determine their analytical performance and the degree of correlation of the test results. A total of 321 samples were characterized based on results from a hemagglutination inhibition assay. The 48 negative and 273 positive samples were used to determine the sensitivity and specificity of the assays. When equivocal results were interpreted as reactive, the sensitivity of the immunoassays ranged from 98.9 to 99.9% and the specificity ranged from 77.1 to 95.8%. All assays had positive and negative delta values of less than 2. A significant difference between the mean results of all assays was demonstrated by analysis of variance. However, post hoc analysis showed there was good correlation in the mean results expressed in IU/ml between some of the assays. Our results show the level of standardization between anti-rubella virus IgG immunoassays reporting results expressed as IU/ml has improved since a previous study in 1992, but further improvement is required.

Assessing proficiency of interpretation of rapid human immunodeficiency virus assays in nonlaboratory settings: ensuring quality of testing.

Rapid antibody tests for the detection of human immunodeficiency virus (HIV) offer an effective means of providing a timely result of HIV serostatus to individuals. The increased use of rapid HIV antibody tests outside the laboratory has highlighted the need for new, cost-effective quality assurance methods to be developed for use in nonlaboratory-based and resource-limited settings. Photographed rapid HIV test results were used in a modified external quality assessment scheme to assess the interpretation proficiency and, therefore, to assess the feasibility of using this method as a basis for a quality assessment program for nonlaboratory-based testing. Participants (n = 148), both experienced and inexperienced in the performance and interpretation of rapid HIV testing, interpreted the photographed results of five rapid HIV assays. These were scored according to the degree of technical discordance. Error scores were grouped according to each participant's technical experience. The accuracy of interpretation for four of the five assays was between 80 and 97%, indicating that the photographed results of samples, including those difficult to read or borderline difficult to read, can be used to assess the proficiency of test operators in interpreting results. Participants had greater difficulty in interpreting samples of weak reactivity; this was consistent across the five assays. Experience played an important role in accurate interpretation, with experienced laboratory participants exhibiting greater proficiency (P < 0.05) in interpreting the results of three of the five rapid HIV assays. It was established that photographed results of rapid HIV assays could be interpreted with accuracy and demonstrated that prior experience resulted in a more accurate interpretation performance.

HIV testing in 2006: issues and methods.

Infection with HIV and subsequent development of AIDS is a pandemic. The Joint United Nations Program on HIV/AIDS together with the WHO and many relevant funding bodies demand that those infected should be reliably identified so that people who need, or will need, therapy may be provided for over time. This means that there is a renewed interest in testing for HIV and in laboratories' performances and quality. Whatever the conditions under which testing is performed, and whatever the levels of training, the tests and their outcomes must exhibit equivalent, high standards of performance and reliable results. This is regardless of whether testing is conducted in the most sophisticated laboratories (either diagnostic or transfusion screening) to voluntary testing and counseling centers where those conducting testing may not be technically trained. This is not currently the case, especially in some places where HIV is most prevalent. To achieve uniformly high performance standards, quality assurance programs are imperative, but currently not sufficiently valued to be well supported with adequate funding or human resources. Accurate HIV testing is a cornerstone of blood safety, diagnosis of infection, patient management and surveillance.

Humoral immune response to primary rubella virus infection.

An assay capable of distinguishing between the immune response generated by recent exposure to rubella virus and the immune response existing as a result of past exposure or immunization is required for the diagnosis of primary rubella virus infection, especially in pregnant women. Avidity assays, which are based on the premise that chaotropic agents can be used to selectively dissociate the low-avidity antibodies generated early in the course of infection, have become routinely used in an effort to accomplish this. We have thoroughly investigated the immunological basis of an avidity assay using a viral lysate-based assay and an enzyme-linked immunosorbent assay (ELISA) based on a peptide analogue of the putative immunodominant region of the E1 glycoprotein (E1208-239). The relative affinities of the antibodies directed against E1208-239 were measured by surface plasmon resonance and were found to correlate well with the avidity index calculated from the ELISA results. We found that the immune response generated during primary rubella virus infection consists of an initial low-affinity peak of immunoglobulin M (IgM) reactivity followed by transient peaks of low-avidity IgG3 and IgA reactivity. The predominant response is an IgG1 response which increases in concentration and affinity progressively over the course of infection. Incubation with the chaotropic agent used in the avidity assay abolished the detection of the early low-affinity peaks of IgM, IgA, and IgG3 reactivity while leaving the high-affinity IgG1 response relatively unaffected. The present study supported the premise that avidity assays based on appropriate antigens can be useful to confirm primary rubella virus infection.

Commercial enzyme immunoassay adapted for the detection of antibodies to hepatitis C virus in dried blood spots.

BACKGROUND: Dried blood spots (DBS) provide a convenient method for blood sample collection in many settings where the prevalence of infection with hepatitis C virus (HCV) is increasing. Consequently, HCV assays are required that produce reliable results using samples derived from DBS. OBJECTIVES AND STUDY DESIGN: The optimum buffer for the elution of samples from DBS was selected and the performance of a commercial enzyme immunoassay (EIA) was evaluated using these DBS eluates and paired plasma samples. RESULTS: DBS with paired plasma samples were compared using this modified commercial EIA, which was found to have an estimated sensitivity and specificity of approximately 100% for detecting anti-HCV antibodies in DBS. CONCLUSION: A DBS-based assay for the detection of antibodies to HCV will prove valuable for collecting epidemiological data in the field or in under resourced settings.

Evaluation of three immunoassays used for detection of anti-rubella virus immunoglobulin M antibodies.

Three automated assays (Abbott AxSYM, Bayer ADVIA Centaur, and bioMerieux VIDAS) used for the detection of rubella virus-specific immunoglobulin M were evaluated. A total of 57 samples from individuals with evidence of infection with rubella virus were used to estimate sensitivity, and 220 samples from blood donors and individuals attending an antenatal clinic who had no evidence of recent infection were used to estimate specificity. Seroconversion panels comprising an additional 31 samples from four individuals were used to determine clinical sensitivity. Samples containing potentially cross-reacting substances were also tested. The sensitivities of the three assays ranged from 84.2 to 96.5%, and the specificities ranged from 96.8 to 99.9%. The Abbott AxSYM assay detected more reactive samples than the other two assays when a panel of 57 positive samples was tested. Bayer ADVIA Centaur detected more reactive samples in the seroconversion panels than the other two assays. All three assays evaluated reported a reactive result in 1 or more of the 48 samples containing potentially cross-reacting analytes. The assays demonstrated comparable performance in testing of a well-characterized panel of samples.

Incidence immunoassay for distinguishing recent from established HIV-1 infection in therapy-naive populations.

OBJECTIVE: To identify a specific marker of recent HIV-1 infection. DESIGN: The humoral immune response in individuals recently infected with HIV-1 was followed by analysing the antibody isotype-specific response generated to HIV-1 antigens in sequential samples collected during and following seroconversion. METHODS: Antibody isotype-specific HIV-1 Western blots were analysed to identify interactions indicative of recent HIV-1 infection. These responses were further quantified using an antibody isotype-specific enzyme-linked immunoabsorbent assay based on recombinant HIV-1 antigens. RESULTS: During maturation of the immune response to HIV-1 infection, a rapid and enduring IgG1 isotype response was seen to all the major proteins transcribed by env, gag and pol. An early transient peak of IgG3 reactivity to p24 was observed over an interval of approximately 1-4 months following HIV-1 infection. The presence of IgG3 reactivity to p24 permitted established infection to be distinguished from recently infected individuals during this time period. CONCLUSION: An assay for anti-p24 IgG3 reactivity would provide an estimate of the incidence of HIV infection that may be applicable for epidemiological surveys as well as for monitoring new infections during vaccine trials and for managing treatment programmes.

Validation of assembled nucleic acid-based tests in diagnostic microbiology laboratories.

Medical microbiology and virology laboratories use nucleic acid tests (NAT) to detect genomic material of infectious organisms in clinical samples. Laboratories choose to perform assembled (or in-house) NAT if commercial assays are not available or if assembled NAT are more economical or accurate. One reason commercial assays are more expensive is because extensive validation is necessary before the kit is marketed, as manufacturers must accept liability for the performance of their assays, assuming their instructions are followed. On the other hand, it is a particular laboratory's responsibility to validate an assembled NAT prior to using it for testing and reporting results on human samples. There are few published guidelines for the validation of assembled NAT. One procedure that laboratories can use to establish a validation process for an assay is detailed in this document. Before validating a method, laboratories must optimise it and then document the protocol. All instruments must be calibrated and maintained throughout the testing process. The validation process involves a series of steps including: (i) testing of dilution series of positive samples to determine the limits of detection of the assay and their linearity over concentrations to be measured in quantitative NAT; (ii) establishing the day-to-day variation of the assay's performance; (iii) evaluating the sensitivity and specificity of the assay as far as practicable, along with the extent of cross-reactivity with other genomic material; and (iv) assuring the quality of assembled assays using quality control procedures that monitor the performance of reagent batches before introducing new lots of reagent for testing.

Advances in laboratory testing for HIV.

In 2004, the diagnosis of established human immunodeficiency virus (HIV) infection can be made with close to 100% assurity. The extraordinarily engineered performances of HIV-screening assays are unprecedented. The well-established confirmatory tests performed by well-versed laboratories using criteria that are well understood in order to interpret the results of these tests give highly accurate outcomes of diagnostic testing strategies. Furthermore, the ability to monitor the progress of the infection and the viral pathogenesis is possible through the use of tests that quantify viral load or the peripheral CD4+ T-cells and other lymphocyte sub-type levels. Newer laboratory testing mechanisms, such as assessment of reverse transcriptase activity and sophisticated cell staining and flow cytometric analyses, have been used to map disease processes and progress on a research level and may be used in future to fine-tune therapy and to follow disease progression in even greater detail. Regulation of all HIV tests is of the highest level in Australia. In-house tests will be expected to conform to the levels specified for commercially produced tests.

External quality assessment program for Chlamydia trachomatis diagnostic testing by nucleic acid amplification assays.

We report the results from 57 Australian diagnostic laboratories testing two external quality assessment panels using either the Roche Amplicor Chlamydia trachomatis test (R-PCR) or the Abbott LCx Chlamydia trachomatis assay (A-ligase chain reaction [LCR]). Panel samples were either normal urine spiked with Chlamydia trachomatis antigen or clinical urine specimens. There was no significant difference between laboratories or between assays in detection of C. trachomatis-positive clinical samples. Only at the lower limit of detection of the assays did the R-PCR demonstrate increased sensitivity over the A-LCR in the detection of C. trachomatis antigen. However, it was found that single-sample testing could lead to decreased test sensitivity. Detection of the presence of inhibitors of nucleic acid amplification differed between laboratories.

Sensitivity of HCV RNA and HIV RNA blood screening assays.

BACKGROUND: The FDA requirement for sensitivity of viral NAT methods used in blood screening is a 95-percent detection limit of 100 copies per mL, whereas the NAT screening system should have a sensitivity of at least 5000 copies per mL per individual donation. According to the Common Technical Specifications of the European Directive 98/79/EC for in vitro diagnostics, viral standard dilutions (calibrated against the WHO standard) should be tested at least 24 times for a statistically valid assessment of the 95-percent detection limit. STUDY DESIGN AND METHODS: Viral standard dilution panels (PeliCheck, VQC-CLB) were prepared for HCV RNA genotypes 1 and 3 and for HIV RNA genotypes B and E. In a multicenter study, 23 laboratories tested the panels all together in 8 to 91 test runs per NAT method. RESULTS: The following 95-percent detection limits (and 95% CIs) were found on the HCV RNA genotype 1 reference panels (shown as geq/mL): Gen-Probe TMA, 85 (64-118); AmpliScreen, 126 (83-225); AmpliScreen with NucliSens Extractor, 21 (13-44); Amplicor with NucliSens Extractor, 69 (50-102), and Amplicor with Qiagen extraction technology, 144 (74-102). On HIV RNA genotype B dilution panels, the following 95-percent detection limits were found (shown as geq/mL): Gen-Probe TMA, 31 (20-52); AmpliScreen, 126 (67-311); AmpliScreen with NucliSens Extractor, 37 (23-69), and NucliSens QL assay, 123 (51-566). HIV RNA genotype E panels were detected with equal sensitivity as HIV RNA genotype B panels. In the Gen-Probe TMA assay, the 50-percent detection limits on HIV RNA type B and type E were 3.6 (2.6-5.0) and 3.9 (2.4-5.8) geq per mL, respectively. The HCV RNA genotype 1 and 3 standards were detected with equal sensitivity. CONCLUSION: The differences in sensitivity between NAT assays can be explained by the input of isolated viral nucleic acid in the amplification reactions. The FDA requirements for sensitivity of NAT blood screening assays can be met by the Gen-probe TMA, as well as by the AmpliScreen assays, particularly when combined with the NucliSens Extractor.