Quality control and external quality assessment for the independent clinic-based evaluation of point-of-care testing to detect Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis in eight countries.

BACKGROUND: Sexually transmitted infections caused by Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Trichomonas vaginalis (TV) remain significant global health problems. The World Health Organization (WHO) has recently conducted a multi-faceted, multi-country validation study (ProSPeRo), which included an evaluation of the Xpert CT/NG and Xpert TV assays on the GeneXpert system (Cepheid, Sunnyvale, Ca., USA) in clinic-based settings across eight countries. To support the study, a training and quality management system was implemented and evaluated. METHODS: A comprehensive training program for the study was developed. Quality control (QC) and external quality assessment (EQA) samples were provided by an accredited quality assurance provider. QC testing was conducted at 14 point-of-care testing (POCT) clinics, while EQA samples were tested by the POCT sites and a reference laboratory supporting each clinic. RESULTS: For QC testing, concordance with the expected results for CT and NG was > 99% and rates of unsuccessful tests were < 4%. For TV testing, concordance was similar (97%), but rates of unsuccessful tests were high (18%), particularly in the 'TV negative' sample. For EQA testing initially conducted in 2018, concordance was 100% for CT and NG, and 90% for TV for the reference laboratory group (which used non-GeneXpert systems). Concordance for the POCT group was also high (> 94%) for all analytes, but this cohort (which used GeneXpert systems) exhibited a high rate of unsuccessful TV tests. All but one of these unsuccessful tests was subcategorised as 'invalid'. CONCLUSIONS: The high level of concordance for QC and EQA testing confirm that the trained operators at the POC clinical sites were competent to conduct POC testing and that the training and quality systems implemented for the ProSPeRo study were effective. The quality materials used were satisfactory for CT and NG but exhibited poor performance for TV testing on the GeneXpert system. The WHO should continue to work with industry and EQA providers to provide improved materials that are reliable, stable and cost effective for quality management, as it seeks to rollout molecular-based STI POCT in non-laboratory-based settings. TRIAL REGISTRATION: Ethics approval to conduct the ProSPeRo study was granted by the WHO Ethics Review Committee.

Comprehensive, comparative evaluation of 25 automated SARS-CoV-2 serology assays.

IMPORTANCE: We have previously highlighted the fact that hundreds of SARS-CoV-2 serology tests were released months after the onset of the COVID-19 pandemic. Of the hundreds of studies investigating the test kits' performance, few were comparative reports, using the same comprehensive sample set across multiple tests. Recently, we reported a comparative assessment of 35 rapid diagnostic tests (RDTs) or microtiter plate enzyme immunoassays (EIA) for use in low- and middle-income countries, using a large sample set from individuals with a history of COVID-19. Only a few tests meet WHO Target Product Profile performance requirements. This study reports on the performance of a further 25 automated SARS-CoV-2 immunoassays using the same panel of samples. The results highlight the better analytical and clinical performance of automated serology test kits compared with RDTs, and the importance of independent comparative assessments to inform the use and procurement of these tests for both diagnostic and epidemiological investigations.

Comprehensive, Comparative Evaluation of 35 Manual SARS-CoV-2 Serological Assays.

The onset of the coronavirus disease 2019 (COVID-19) pandemic resulted in hundreds of in vitro diagnostic devices (IVDs) coming to market, facilitated by regulatory authorities allowing "emergency use" without a comprehensive evaluation of performance. The World Health Organization (WHO) released target product profiles (TPPs) specifying acceptable performance characteristics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) assay devices. We evaluated 26 rapid diagnostic tests and 9 enzyme immunoassays (EIAs) for anti-SARS-CoV-2, suitable for use in low- and middle-income countries (LMICs), against these TPPs and other performance characteristics. The sensitivity and specificity ranged from 60.1 to 100% and 56.0 to 100%, respectively. Five of 35 test kits reported no false reactivity for 55 samples with potentially cross-reacting substances. Six test kits reported no false reactivity for 35 samples containing interfering substances, and only one test reported no false reactivity with samples positive for other coronaviruses (not SARS-CoV-2). This study demonstrates that a comprehensive evaluation of the performance of test kits against defined specifications is essential for the selection of test kits, especially in a pandemic setting. IMPORTANCE The markets have been flooded with hundreds of SARS-CoV-2 serology tests, and although there are many published reports on their performance, comparative reports are far fewer and tend to be limited to only a few tests. In this report, we comparatively assessed 35 rapid diagnostic tests or microtiter plate enzyme immunoassays (EIAs) using a large set of samples from individuals with a history of mild to moderate COVID-19, commensurate with the target population for serosurveillance, which included serum samples from individuals previously infected, at undetermined time periods, with other seasonal human coronaviruses, Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-1. The significant heterogeneity in their performances, with only a few tests meeting WHO target product profile performance requirements, highlights the importance of independent comparative assessments to inform the use and procurement of these tests for both diagnostics and epidemiological investigations.

Time to address quality control processes applied to antibody testing for infectious diseases.

As testing for infectious diseases moves from manual, biological testing such as complement fixation to high throughput automated autoanalyzer, the methods for controlling these assays have also changed to reflect those used in clinical chemistry. However, there are many differences between infectious disease serology and clinical chemistry testing, and these differences have not been considered when applying traditional quality control methods to serology. Infectious disease serology, which is highly regulated, detects antibodies of varying classes and to multiple and different antigens that change according to the organisms' genotype/serotype and stage of disease. Although the tests report a numerical value (usually signal to cut-off), they are not measuring an amount of antibodies, but the intensity of binding within the test system. All serology assays experience lot-to-lot variation, making the use of quality control methods used in clinical chemistry inappropriate. In many jurisdictions, the use of the manufacturer-provided kit controls is mandatory to validate the test run. Use of third-party controls, which are highly recommended by ISO 15189 and the World Health Organization, must be manufactured in a manner whereby they have minimal lot-to-lot variation and at a level where they detect exceptional variation. This paper outlines the differences between clinical chemistry and infectious disease serology and offers a range of recommendations when addressing the quality control of infectious disease serology.

Implementation of Novel Quality Assurance Program for Hepatitis C Viral Load Point of Care Testing.

All patients should have access to accurate and timely test results. The introduction of point of care testing (PoCT) for infectious diseases has facilitated access to those unable to access traditional laboratory-based medical testing, including those living in remote and regional locations, or individuals who are marginalized or incarcerated individuals. In many countries, laboratory testing for infectious diseases, such as hepatitis C virus (HCV), is performed in a highly regulated environment. However, this is not the case for PoCT, where testing is performed by non-laboratory staff and quality controls are often lacking. An assessment of the provision of laboratory-based quality assurance to PoCT for infectious disease was conducted and the barriers to participation identified. A novel approach to providing quality assurance to PoCT sites, in particular those testing for HCV, was designed and piloted. This novel approach incudes identifying and validating sample types that are inactivated and stable at ambient temperature, creating cost-effective supply chains to facilitate logistics of samples, and the development of a smart phone-enabled portal for data entry and analyses. The creation and validation of this approach to quality assurance of PoCT removes the barriers to participation and acts to improve the quality and accuracy of testing, reduce errors and waste, and improve patient outcomes.

Low Genetic Diversity of Hepatitis B Virus Surface Gene amongst Australian Blood Donors.

Variants in the small surface gene of hepatitis B virus (HBV), which codes for viral surface antigen (HBsAg), can affect the efficacy of HBsAg screening assays and can be associated with occult HBV infection (OBI). This study aimed to characterise the molecular diversity of the HBV small surface gene from HBV-reactive Australian blood donors. HBV isolates from 16 HBsAg-positive Australian blood donors' plasma were sequenced and genotyped by phylogenies of viral coding genes and/or whole genomes. An analysis of the genetic diversity of eight HBV small surface genes from our 16 samples was conducted and compared with HBV sequences from NCBI of 164 international (non-Australian) blood donors. Genotypes A-D were identified in our samples. The region of HBV small surface gene that contained the sequence encoding the 'a' determinant had a greater genetic diversity than the remaining part of the gene. No escape mutants or OBI-related variants were observed in our samples. Variant call analysis revealed two samples with a nucleotide deletion leading to truncation of polymerase and/or large/middle surface amino acid sequences. Overall, we found that HBV small surface gene sequences from Australian donors demonstrated a lower level of genetic diversity than those from non-Australian donor population included in the study.

The Standardization and Control of Serology and Nucleic Acid Testing for Infectious Diseases.

Historically, the detection of antibodies against infectious disease agents was achieved using test systems that utilized biological functions such as neutralization, complement fixation, hemagglutination, or visualization of binding of antibodies to specific antigens, by testing doubling dilutions of the patient sample to determine an endpoint. These test systems have since been replaced by automated platforms, many of which have been integrated into general medical pathology. Methods employed to standardize and control clinical chemistry testing have been applied to these serology tests. However, there is evidence that these methods are not suitable for infectious disease serology. An overriding reason is that, unlike testing for an inert chemical, testing for specific antibodies to infectious disease agents is highly variable; the measurand for each test system varies in choice of antigen, antibody classes/subclasses, modes of detection, and assay kinetics, and individuals' immune responses vary with time after exposure, individual immune-competency, nutrition, treatment, and exposure to variable circulating sero- or genotypes or organism mutations. Therefore, unlike that of inert chemicals, the quantification of antibodies cannot be standardized using traditional methods. However, there is evidence that the quantification of nucleic acid testing, reporting results in international units, has been successful across many viral load tests. Similarly, traditional methods used to control clinical chemistry testing, such as Westgard rules, are not appropriate for serology testing for infectious diseases, mainly due to variability due to frequent reagent lot changes. This review investigates the reasons why standardization and control of infectious diseases should be further investigated and more appropriate guidelines should be implemented.

Real-world monitoring progress towards the elimination of hepatitis C virus in Australia using sentinel surveillance of primary care clinics; an ecological study of hepatitis C virus antibody tests from 2009 to 2019.

To achieve the elimination of the hepatitis C virus (HCV), sustained and sufficient levels of HCV testing is critical. The purpose of this study was to assess trends in testing and evaluate the effectiveness of strategies to diagnose people living with HCV. Data were from 12 primary care clinics in Victoria, Australia, that provide targeted services to people who inject drugs (PWID), alongside general health care. This ecological study spanned 2009-2019 and included analyses of trends in annual numbers of HCV antibody tests among individuals with no previous positive HCV antibody test recorded and annual test yield (positive HCV antibody tests/all HCV antibody tests). Generalised linear models estimated the association between count outcomes (HCV antibody tests and positive HCV antibody tests) and time, and χ2 test assessed the trend in test yield. A total of 44 889 HCV antibody tests were conducted 2009-2019; test numbers increased 6% annually on average [95% confidence interval (CI) 4-9]. Test yield declined from 2009 (21%) to 2019 (9%) (χ2P = <0.01). In more recent years (2013-2019) annual test yield remained relatively stable. Modest increases in HCV antibody testing and stable but high test yield within clinics delivering services to PWID highlights testing strategies are resulting in people are being diagnosed however further increases in the testing of people at risk of HCV or living with HCV may be needed to reach Australia's HCV elimination goals.

Validation of Dried Tube Sample Format Quality Controls for the Monitoring of Viral Load and Blood Screening Assays.

HIV viral load (VL) and donor screening assays experience variation and require quaity assurance (QA). NRL sought to confirm a dried tube sample format (HIVDTS) sample type for use in quality control (QC) programs for HIV molecular testing. 50 µL of HIV supernatant at 1 × 105 copies per millilitre (copies/mL)) was dried for 48 hours at room temperature. Post-production and shipped integrity studies were undertaken. Dried HIVDTS was reconstituted in PBS buffer and tested in HIV VL (six participants) or blood screening assays (four participants). Results were entered into NRL's QC monitoring software (EDCNet™) for analysis. The mean of 224 VL results when HIVDTS QCs were tested in Biocentric HIV GENERIC Charge Virale assay was 4.54 log10 copies/mL, with the percentage coefficient of variation (CV%) ranging from 1.75 to 13.20%. The mean Ct value for HIVDTS QCs tested on Roche Cobas MPX assay results was 28.71 (range 28.33 to 29.14), with CV% ranging from 1.56 to 3.98%. The study confirms HIVDTS QCs can effectively monitor the performance of HIV molecular testing and offers a cheaper alternative to commercial QC samples that require cold-chain shipping on dry ice and UN3373 conditions.

Improving the coverage and accuracy of syphilis testing: The development of a novel rapid, point-of-care test for confirmatory testing of active syphilis infection and its early evaluation in China and South Africa.

BACKGROUND: Current point-of-care tests (POCT) for syphilis, based on the detection of Treponema pallidum (TP) total antibodies, have limited capacity in distinguishing between active and past/treated syphilis. We report the development and early evaluation of a new prototype POCT based on the detection of TP-IgA antibodies, a novel biomarker for active syphilis. METHODS: The TP-IgA POCT (index test) was developed in response to the World Health Organisation (WHO) target product profile (TPP) for a POCT for confirmatory syphilis testing. Two sub-studies were conducted consecutively using 458 pre-characterised stored plasma samples in China (sub-study one, addressing the criteria for the WHO TPP), and 503 venous blood samples collected from pregnant/postpartum women in South Africa (sub-study two, addressing potential clinical utility). Performance of the index test was assessed against standard laboratory-based serology using a combination of treponemal (TPHA) and non-treponemal (rapid plasma reagin [RPR]) tests. FINDINGS: In sub-study one, the index test demonstrated 96·1% (95%CI=91·7%-98·5%) sensitivity and 84·7% (95%CI=80·15-88·6%) specificity for identification of active syphilis (TPHA positive, RPR positive). It correctly identified 71% (107/150) samples of past-treated syphilis (TPHA positive, RPR negative). In sub-study two, the index test achieved 100% (95%CI=59%-100%) sensitivity for active syphilis and correctly identified all nine women with past syphilis. INTERPRETATION: The TP-IgA POCT has met the WHO TPP for a POCT for diagnosis of active syphilis and demonstrated its potential utility in a clinical setting. Future studies are warranted to evaluate field performance of the final manufactured test. FUNDING: Saving Lives at Birth: Grand Challenge for Development, Thrasher Research Fund, and the Victorian Government Operational Infrastructure Scheme.

Does a change in quality control results influence the sensitivity of an anti-HCV test?

Background Laboratories use quality control (QC) testing to monitor the extent of normal variation. Assay lot number changes contribute the greatest amount of variation in infectious disease serology testing. An unexpected change in six lots of an anti-HCV assay allowed the determination of the effect these lot changes made to the assay's clinical sensitivity. Methods Two sets of seroconversion samples comprising of 44 individual samples and 9 external quality assessment scheme (EQAS) samples, all positive to anti-HCV, were tested in affected and unaffected assay lots, and the difference in the quantitative and qualitative results of the samples was analyzed. Results Of 44 low-positive seroconversion samples tested in affected and unaffected assay lots, only three samples had results reported below the assay cutoff when tested on two of the six affected assay lot. A further sample had results below the cutoff for only one affected lot. None of the EQAS samples reported false-negative results. Samples having a signal to cutoff value of less than 6.0 generally had lower results in the affected lots compared with the unaffected lots. Conclusions Unexpected changes in QC reactivity related to variation, in particular assay lot changes, may affect patient results. This study demonstrated that QConnect Limits facilitated the detection of an unexpectedly large variation in QC test results, allowed for the identification of the root cause of the change, and showed that the risk associated with the change was low but credible. The use of evidence-based QC program is essential to detect changes in test systems.

WHO international standard for anti-rubella: learning from its application.

The WHO international standard for anti-rubella was first established in the 1960s when clinical diagnostics were in their infancy. Since the endorsement of the first international standard for anti-rubella IgG (RUBI-1-94), new rubella vaccines have been developed and global coverage of rubella vaccination has increased. Methods used to measure concentrations of anti-rubella IgG have also evolved to rapid, high-throughput binding assays, which have replaced often cumbersome and highly technical functional assays. During this timeframe, the protective concentration of antibody was set at 10 IU/mL by extrapolation of functional assay correlates; however, the subpopulation of antibodies within a polyclonal serum that confer protection remained undefined. Anti-rubella assays have variable formats, including antigens used, such that the same clinical sample tested on different assays can report different values with potentially devastating consequences, such as recommending to terminate pregnancy. WHO convened a meeting of experts in the rubella field to discuss the use of RUBI-1-94 and the potential future role of this international standard. The main conclusions of this meeting questioned the appropriateness of 10 IU/mL as the cutoff for protection and acknowledged the continuing role of RUBI-1-94 as a reference preparation to address analytical sensitivity and assay variation.

Tracking the uptake of outcomes of hepatitis B virus testing using laboratory data in Victoria, 2011-16: a population-level cohort study.

Background A priority area in the 2016 Victorian Hepatitis B Strategy is to increase diagnostic testing. This study describes hepatitis B testing and positivity trends in Victoria between 2011 and 2016 using data from a national laboratory sentinel surveillance system. METHODS: Line-listed diagnostic and monitoring hepatitis B testing data among Victorian individuals were collated from six laboratories participating in the Australian Collaboration for Coordinated Enhanced Sentinel Surveillance (ACCESS) of sexually transmissible infections and blood-borne viruses. Diagnostic tests included hepatitis B surface antigen (HBsAg)-only tests and guideline-based hepatitis B tests (defined as a single test event for HBsAg, hepatitis B surface antibody and hepatitis B core antibody). Using available data, the outcomes of testing and/or infection were further classified. Measures reported include the total number of HBsAg and guideline-based tests conducted and the proportion positive, classified as either HBsAg positive or chronic hepatitis B infection. RESULTS: The number of HBsAg tests decreased slightly each year between 2011 and 2016 (from 91043 in 2011 to 79664 in 2016; P < 0.001), whereas the number of guideline-based hepatitis B tests increased (from 8732 in 2011 to 16085 in 2016; P <0.001). The proportion of individuals classified as having chronic infection decreased from 25% in 2011 to 7% in 2016, whereas the proportion classified as susceptible and immune due to vaccination increased (from 29% to 39%, and from 27% to 34%, respectively; P < 0.001). CONCLUSIONS: The study findings indicate an increased uptake of guideline-based hepatitis B testing. The ongoing collection of testing data can help monitor progress towards implementation of the Victorian Hepatitis B Strategy.

Pathology Laboratory Surveillance in the Australian Collaboration for Coordinated Enhanced Sentinel Surveillance of Sexually Transmitted Infections and Blood-Borne Viruses: Protocol for a Cohort Study.

BACKGROUND: Passive surveillance is the principal method of sexually transmitted infection (STI) and blood-borne virus (BBV) surveillance in Australia whereby positive cases of select STIs and BBVs are notified to the state and territory health departments. A major limitation of passive surveillance is that it only collects information on positive cases and notifications are heavily dependent on testing patterns. Denominator testing data are important in the interpretation of notifications. OBJECTIVE: The aim of this study is to establish a national pathology laboratory surveillance system, part of a larger national sentinel surveillance system called ACCESS (the Australian Collaboration for Coordinated Enhanced Sentinel Surveillance). ACCESS is designed to utilize denominator testing data to understand trends in case reporting and monitor the uptake and outcomes of testing for STIs and BBVs. METHODS: ACCESS involves a range of clinical sites and pathology laboratories, each with a separate method of recruitment, data extraction, and data processing. This paper includes pathology laboratory sites only. First established in 2007 for chlamydia only, ACCESS expanded in 2012 to capture all diagnostic and clinical monitoring tests for STIs and BBVs, initially from pathology laboratories in New South Wales and Victoria, Australia, to at least one public and one private pathology laboratory in all Australian states and territories in 2016. The pathology laboratory sentinel surveillance system incorporates a longitudinal cohort design whereby all diagnostic and clinical monitoring tests for STIs and BBVs are collated from participating pathology laboratories in a line-listed format. An anonymous, unique identifier will be created to link patient data within and between participating pathology laboratory databases and to clinical services databases. Using electronically extracted, line-listed data, several indicators for each STI and BBV can be calculated, including the number of tests, unique number of individuals tested and retested, test yield, positivity, and incidence. RESULTS: To date, over 20 million STI and BBV laboratory test records have been extracted for analysis for surveillance monitoring nationally. Recruitment of laboratories is ongoing to ensure appropriate coverage for each state and territory; reporting of indicators will occur in 2019 with publication to follow. CONCLUSIONS: The ACCESS pathology laboratory sentinel surveillance network is a unique surveillance system that collects data on diagnostic testing, management, and care for and of STIs and BBVs. It complements the ACCESS clinical network and enhances Australia's capacity to respond to STIs and BBVs. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/13625.

Monitoring the Control of Sexually Transmissible Infections and Blood-Borne Viruses: Protocol for the Australian Collaboration for Coordinated Enhanced Sentinel Surveillance (ACCESS).

BACKGROUND: New biomedical prevention interventions make the control or elimination of some blood-borne viruses (BBVs) and sexually transmissible infections (STIs) increasingly feasible. In response, the World Health Organization and governments around the world have established elimination targets and associated timelines. To monitor progress toward such targets, enhanced systems of data collection are required. This paper describes the Australian Collaboration for Coordinated Enhanced Sentinel Surveillance (ACCESS). OBJECTIVE: This study aims to establish a national surveillance network designed to monitor public health outcomes and evaluate the impact of strategies aimed at controlling BBVs and STIs. METHODS: ACCESS is a sentinel surveillance system comprising health services (sexual health clinics, general practice clinics, drug and alcohol services, community-led testing services, and hospital outpatient clinics) and pathology laboratories in each of Australia's 8 states and territories. Scoping was undertaken in each jurisdiction to identify sites that provide a significant volume of testing or management of BBVs or STIs or to see populations with particular risks for these infections ("priority populations"). Nationally, we identified 115 health services and 24 pathology laboratories as relevant to BBVs or STIs; purposive sampling was undertaken. As of March 2018, we had recruited 92.0% (104/113) of health services and 71% (17/24) of laboratories among those identified as relevant to ACCESS. ACCESS is based on the regular and automated extraction of deidentified patient data using specialized software called GRHANITE, which creates an anonymous unique identifier from patient details. This identifier allows anonymous linkage between and within participating sites, creating a national cohort to facilitate epidemiological monitoring and the evaluation of clinical and public health interventions. RESULTS: Between 2009 and 2017, 1,171,658 individual patients attended a health service participating in ACCESS network comprising 7,992,241 consultations. Regarding those with unique BBV and STI-related health needs, ACCESS captured data on 366,441 young heterosexuals, 96,985 gay and bisexual men, and 21,598 people living with HIV. CONCLUSIONS: ACCESS is a unique system with the ability to track efforts to control STIs and BBVs-including through the calculation of powerful epidemiological indicators-by identifying response gaps and facilitating the evaluation of programs and interventions. By anonymously linking patients between and within services and over time, ACCESS has exciting potential as a research and evaluation platform. Establishing a national health surveillance system requires close partnerships across the research, government, community, health, and technology sectors. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/11028.

The new screening program to prevent cervical cancer using HPV DNA: getting the balance right in maintaining quality.

Along with the reduction in human papillomavirus (HPV) infection and cervical abnormalities as a result of the successful HPV vaccination program, Australia is adopting a new screening strategy. This involves a new paradigm moving from cervical cytological screening to molecular nucleic acid technology (NAT), using HPV DNA assays as primary screening methodology for cervical cancer prevention. These assays must strike a balance between sufficient clinical sensitivity to detect or predict high-grade cervical intraepithelial lesions, the precursor to cervical cancer, without being too sensitive and detecting transient infection not destined for disease. Ensuring the highest quality HPV NAT is thus a priority in order to reduce the possibility of falsely negative screens and manage the risk associated with false positive HPV NAT test results. How to do this needs informed discussion and on-going refinement of the screening algorithm. This is of relevance as more countries move to more sensitive HPV NAT tests for secondary prevention of cervical cancer and as more HPV assays become available.