Retrospective Assessment of a National Reflex Cryptococcal Antigen Screening Program in South Africa Through Interlaboratory Comparison of Lateral Flow Assay Results.

OBJECTIVE: Reflex cryptococcal antigen (CrAg) screening of blood specimens with a CD4 count of <100 cells/µL was performed at 45 South African CD4 laboratories using a lateral flow assay (LFA). Our objective was to evaluate the reliability of routine LFA results through comparative interlaboratory testing. METHODS: All CrAg-positive and a selected number of CrAg-negative samples from the CD4 laboratories were retested at paired microbiology laboratories using the same LFA. Samples with discordant results were tested at a reference laboratory, using the LFA (with CrAg titers). RESULTS: During interlaboratory testing, 12,502 samples were retested, with 93 (0.7%) discordant results and a between-laboratory agreement of 99.3% (Cohen's kappa, 0.98). The proportion of retested samples with discordant results ranged from 0.17% to 5.31% per laboratory pair (median 0.28%), with 3 reporting >3% of results as discordant. CONCLUSION: Routine CrAg screening results were reliable, with <1% of samples having discordant results, mainly due to interpretation and transcription errors.

Establishing the cost of Xpert MTB/RIF mobile testing in high-burden peri-mining communities in South Africa.

BACKGROUND: Globally, tuberculosis remains a major cause of mortality, with an estimated 1.3 million deaths per annum. The Xpert MTB/RIF assay is used as the initial diagnostic test in the tuberculosis diagnostic algorithm. To extend the national tuberculosis testing programme in South Africa, mobile units fitted with the GeneXpert equipment were introduced to high-burden peri-mining communities. OBJECTIVE: This study sought to assess the cost of mobile testing compared to traditional laboratory-based testing in a peri-mining community setting. METHODS: Actual cost data for mobile and laboratory-based Xpert MTB/RIF testing from 2018 were analysed using a bottom-up ingredients-based approach to establish the annual equivalent cost and the cost per result. Historical cost data were obtained from supplier quotations and the local enterprise resource planning system. Costs were obtained in rand and reported in United States dollars (USD). RESULTS: The mobile units performed 4866 tests with an overall cost per result of $49.16. Staffing accounted for 30.7% of this cost, while reagents and laboratory equipment accounted for 20.7% and 20.8%. The cost per result of traditional laboratory-based testing was $15.44 US dollars (USD). The cost for identifying a tuberculosis-positive result using mobile testing was $439.58 USD per case, compared to $164.95 USD with laboratory-based testing. CONCLUSION: Mobile testing is substantially more expensive than traditional laboratory services but offers benefits for rapid tuberculosis case detection and same-day antiretroviral therapy initiation. Mobile tuberculosis testing should however be reserved for high-burden communities with limited access to laboratory testing where immediate intervention can benefit patient outcomes.

Timely delivery of laboratory efficiency information, Part I: Developing an interactive turnaround time dashboard at a high-volume laboratory.

BACKGROUND: Mean turn-around time (TAT) reporting for testing laboratories in a national network is typically static and not immediately available for meaningful corrective action and does not allow for test-by-test or site-by-site interrogation of individual laboratory performance. OBJECTIVE: The aim of this study was to develop an easy-to-use, visual dashboard to report interactive graphical TAT data to provide a weekly snapshot of TAT efficiency. METHODS: An interactive dashboard was developed by staff from the National Priority Programme and Central Data Warehouse of the National Health Laboratory Service, Johannesburg, South Africa, during 2018. Steps required to develop the dashboard were summarised in a flowchart. To illustrate the dashboard, one week of data from a busy laboratory for a specific set of tests was analysed using annual performance plan TAT cut-offs. Data were extracted and prepared to deliver an aggregate extract, with statistical measures provided, including test volumes, global percentage of tests that were within TAT cut-offs and percentile statistics. RESULTS: Nine steps were used to develop the dashboard iteratively with continuous feedback for each step. The data warehouse environment conformed and stored laboratory information system data in two formats: (1) fact and (2) dimension. Queries were developed to generate an aggregate TAT data extract to create the dashboard. The dashboard successfully delivered weekly TAT reports. CONCLUSION: Implementation of a TAT dashboard can successfully enable the delivery of near real-time information and provide a weekly snapshot of efficiency in the form of TAT performance to identify and quantitate bottlenecks in service delivery.

Timely delivery of laboratory efficiency information, Part II: Assessing the impact of a turn-around time dashboard at a high-volume laboratory.

BACKGROUND: In South Africa's National Health Laboratory Service, ad hoc mean turn-around time (TAT) reporting is an important indicator of performance. However, historic static TAT reporting did not assess very long or very short times. An interactive TAT dashboard was developed using the following TAT measures; (1) median, (2) 75th percentile and (3) percentage of within cut-off TAT to allow for improved differentiation of TAT performance. OBJECTIVES: The objective of our study was to demonstrate increased efficiency achieved by using an interactive TAT dashboard. METHODS: A retrospective descriptive study design was used. Creatinine TAT outcomes were reported over 122 weeks from a high-volume laboratory in Gauteng, South Africa. The percentage of within cut-off and 75th percentile TAT were analysed and reported using Microsoft Excel. A focus group session was used to populate a cause and effect diagram. RESULTS: The percentage of within cut-off TAT increased from 10% in week 4 to 90% and higher from week 81. The 75th percentile decreased from 10 hours in week 4 to under 5 h from week 71. Component TAT analysis revealed that the 75th percentile testing was 5 h or longer for weeks 4, 5 and 48. The 75th percentile review TAT ranged from 1 h to 15 h. From week 41, the review TAT was under 1 h. CONCLUSION: Our study demonstrated that the use of an interactive TAT dashboard coupled with good management can dramatically improve TAT and efficiency in a high-volume laboratory.

Categorising specimen referral delays for CD4 testing: How inter-laboratory distances and travel times impact turn-around time across a national laboratory service in South Africa.

BACKGROUND: The South African National Health Laboratory Service provides laboratory services for public sector health facilities, utilising a tiered laboratory model to refer samples for CD4 testing from 255 source laboratories into 43 testing laboratories. OBJECTIVE: The aim of this study was to determine the impact of distance on inter-laboratory referral time for public sector testing in South Africa in 2018. METHODS: A retrospective cross-sectional study design analysed CD4 testing inter-laboratory turn-around time (TAT) data for 2018, that is laboratory-to-laboratory TAT from registration at the source to referral receipt at the testing laboratory. Google Maps was used to calculate inter-laboratory distances and travel times. Distances were categorised into four buckets, with the median and 75th percentile reported. Wilcoxon scores were used to assess significant differences in laboratory-to-laboratory TAT across the four distance categories. RESULTS: CD4 referrals from off-site source laboratories comprised 49% (n = 1 390 510) of national reporting. A positively skewed distribution of laboratory-to-laboratory TAT was noted, with a median travel time of 11 h (interquartile range: 7-17), within the stipulated 12 h target. Inter-laboratory distance categories of less than 100 km, 101-200 km, 201-300 km and more than 300 km (p < 0.0001) had 75th percentiles of 8 h, 17 h, 14 h and 27 h. CONCLUSION: Variability in inter-laboratory TAT was noted for all inter-laboratory distances, especially those exceeding 300 km. The correlation between distance and laboratory-to-laboratory TAT suggests that interventions are required for distant laboratories.

Addressing antiretroviral therapy-related diagnostic coverage gaps across South Africa using a programmatic approach.

BACKGROUND: A major challenge facing South Africa is the concomitant HIV and tuberculosis epidemics. The National Health Laboratory Service provides testing for staging HIV-positive patients, monitoring patients on antiretroviral therapy (ART) and diagnosing tuberculosis. Not all health districts have equivalent ART-related coverage in particular for CD4 and HIV viral load testing. OBJECTIVES: The Integrated Tiered Service Delivery Model coverage precinct approach was used to address ART-related testing service coverage gaps in a manner that balances cost, quality and equity. METHODS: An algorithm was developed to identify and address ART-related diagnostic coverage gaps. Data was extracted from the corporate data warehouse and Oracle systems for the period of April 2015 to March 2016. Daily test volumes were based on 21.73 working days per month. Data were analysed using MS Excel and mapped using ArcCatalog and ArcMap. Capacity analysis was informed by the available testing-platforms. RESULTS: Health district daily HIV viral load volumes ranged from 2 to 1308 samples. Nineteen candidate laboratories were identified to address the coverage gaps. Following the proximity analysis, testing was consolidated at four candidate laboratories, resulting in 13 revised candidate laboratories. The revised candidate laboratory daily HIV viral load referrals ranged between 5 and 205 samples, with CD4 volumes between 6 and 85 samples. Remaining coverage gaps were identified in seven municipalities. CONCLUSIONS: The study demonstrated that the service coverage precinct approach could be used to identify coverage gaps for a defined ART-related testing repertoire.

District and sub-district analysis of cryptococcal antigenaemia prevalence and specimen positivity in KwaZulu-Natal, South Africa.

BACKGROUND: Cryptococcal meningitis (CM) is a leading cause of mortality among HIV-positive South Africans. Reflex cryptococcal antigen (CrAg) testing of remnant plasma was offered as a pilot prior to implementation in October 2016 in KwaZulu-Natal province. The national reflex CrAg positivity was 5.4% compared to 7.3% for KwaZulu-Natal. OBJECTIVES: The aim of this study was to interrogate CrAg positivity by health levels to identify hotspots. METHOD: Data for the period October 2016 to June 2017 were analysed. Health district CrAg positivity and prevalence were calculated, with the latter using de-duplicated patient data. The district CrAg positivity and the number of CrAg-positive specimens per health facility were mapped using ArcGIS. For districts with the highest CrAg positivity, a sub-district CrAg positivity analysis was conducted. RESULTS: The provincial CrAg positivity was 7.6%. District CrAg positivity ranged from 5.7% (Ugu) to 9.6% (Umkhanyakude) with prevalence ranging from 5.5% (Ugu) to 9.7% (Umkhanyakude). The highest CrAg positivity was reported for the Umkhanyakude (9.6%) and King Cetswayo (9.5%) districts. In these two districts, CrAg positivity of 10% was noted in the Umhlabuyalingana (10.0%), Jozini (10.2%), uMhlathuze (10.5%) and Nkandla (10.8%) subdistricts. In these subdistricts, 135 CrAg-positive samples were reported for the Ngwelezane hospital followed by 41 and 43 at the Hlabisa and Manguzi hospitals respectively. CONCLUSION: Cryptococcal antigen positivity was not uniformly distributed at either the district or sub-district levels, with identified facility hotspots in the Umkhanyakude and King Cetswayo districts. This study demonstrates the value of laboratory data to identify hotspots for planning programmatic interventions.

Programmatic implications of implementing the relational algebraic capacitated location (RACL) algorithm outcomes on the allocation of laboratory sites, test volumes, platform distribution and space requirements.

INTRODUCTION: CD4 testing in South Africa is based on an integrated tiered service delivery model that matches testing demand with capacity. The National Health Laboratory Service has predominantly implemented laboratory-based CD4 testing. Coverage gaps, over-/under-capacitation and optimal placement of point-of-care (POC) testing sites need investigation. OBJECTIVES: We assessed the impact of relational algebraic capacitated location (RACL) algorithm outcomes on the allocation of laboratory and POC testing sites. METHODS: The RACL algorithm was developed to allocate laboratories and POC sites to ensure coverage using a set coverage approach for a defined travel time (T). The algorithm was repeated for three scenarios (A: T = 4; B: T = 3; C: T = 2 hours). Drive times for a representative sample of health facility clusters were used to approximate T. Outcomes included allocation of testing sites, Euclidian distances and test volumes. Additional analysis included platform distribution and space requirement assessment. Scenarios were reported as fusion table maps. RESULTS: Scenario A would offer a fully-centralised approach with 15 CD4 laboratories without any POC testing. A significant increase in volumes would result in a four-fold increase at busier laboratories. CD4 laboratories would increase to 41 in scenario B and 61 in scenario C. POC testing would be offered at two sites in scenario B and 20 sites in scenario C. CONCLUSION: The RACL algorithm provides an objective methodology to address coverage gaps through the allocation of CD4 laboratories and POC sites for a given T. The algorithm outcomes need to be assessed in the context of local conditions.

Screening HIV-Infected Patients with Low CD4 Counts for Cryptococcal Antigenemia prior to Initiation of Antiretroviral Therapy: Cost Effectiveness of Alternative Screening Strategies in South Africa.

BACKGROUND: In 2015 South Africa established a national cryptococcal antigenemia (CrAg) screening policy targeted at HIV-infected patients with CD4+ T-lymphocyte (CD4) counts <100 cells/ µl who are not yet on antiretroviral treatment (ART). Two screening strategies are included in national guidelines: reflex screening, where a CrAg test is performed on remnant blood samples from CD4 testing; and provider-initiated screening, where providers order a CrAg test after a patient returns for CD4 test results. The objective of this study was to compare costs and effectiveness of these two screening strategies. METHODS: We developed a decision analytic model to compare reflex and provider-initiated screening in terms of programmatic and health outcomes (number screened, number identified for preemptive treatment, lives saved, and discounted years of life saved) and screening and treatment costs (2015 USD). We estimated a base case with prevalence and other parameters based on data collected during CrAg screening pilot projects integrated into routine HIV care in Gauteng, Free State, and Western Cape Provinces. We conducted sensitivity analyses to explore how results change with underlying parameter assumptions. RESULTS: In the base case, for each 100,000 CD4 tests, the reflex strategy compared to the provider-initiated strategy has higher screening costs ($37,536 higher) but lower treatment costs ($55,165 lower), so overall costs of screening and treatment are $17,629 less with the reflex strategy. The reflex strategy saves more lives (30 lives, 647 additional years of life saved). Sensitivity analyses suggest that reflex screening dominates provider-initiated screening (lower total costs and more lives saved) or saves additional lives for small additional costs (< $125 per life year) across a wide range of conditions (CrAg prevalence, patient and provider behavior, patient survival without treatment, and effectiveness of preemptive fluconazole treatment). CONCLUSIONS: In countries with substantial numbers of people with untreated, advanced HIV disease such as South Africa, CrAg screening before initiation of ART has the potential to reduce cryptococcal meningitis and save lives. Reflex screening compared to provider-initiated screening saves more lives and is likely to be cost saving or have low additional costs per additional year of life saved.

Compliance to HIV treatment monitoring guidelines can reduce laboratory costs.

BACKGROUND: Panel tests are a predetermined group of tests commonly requested together to provide a comprehensive and conclusive diagnosis, for example, liver function test (LFT). South African HIV antiretroviral treatment (ART) guidelines recommend individual tests for toxicity monitoring over panel tests. In 2008, the National Health Laboratory Services (NHLS) request form was redesigned to list individual tests instead of panel tests and removed the 'other tests' box option to facilitate efficient ART laboratory monitoring. OBJECTIVES: This study aimed to demonstrate changes in laboratory expenditure, for individual and panel tests, for ART toxicity monitoring. METHOD: NHLS Corporate Data Warehouse (CDW) data were extracted for HIV conditional grant accounts to assess ART toxicity monitoring laboratory expenditure between 2010/2011 and 2014/2015. Data were classified based on the tests requested, as either panel (LFT or urea and electrolytes) or individual (alanine transaminase or creatinine) tests. RESULTS: Expenditure on panel tests reduced from R340 million in 2010/2011 to R140m by 2014/2015 (reduction of R204m) and individual test expenditure increased from R34m to R76m (twofold increase). A significant reduction in LFT panel expenditure was noted, reducing from R322m in 2010/2011 to R130m in 2014/2015 (60% reduction). CONCLUSION: Changes in toxicity monitoring guidelines and the re-engineering of the NHLS request form successfully reduced expenditure on panel tests relative to individual tests. The introduction of order entry systems could further reduce unnecessary laboratory expenditure.

Compliance to HIV Treatment Monitoring Guidelines can Reduce Laboratory Costs

Background: Panel tests are a predetermined group of tests commonly requested together to provide a comprehensive and conclusive diagnosis; for example; liver function test (LFT). South African HIV antiretroviral treatment (ART) guidelines recommend individual tests for toxicity monitoring over panel tests. In 2008; the National Health Laboratory Services (NHLS) request form was redesigned to list individual tests instead of panel tests and removed the 'other tests' box option to facilitate efficient ART laboratory monitoring.Objectives: This study aimed to demonstrate changes in laboratory expenditure; for individual and panel tests; for ART toxicity monitoring.Method: NHLS Corporate Data Warehouse (CDW) data were extracted for HIV conditional grant accounts to assess ART toxicity monitoring laboratory expenditure between 2010/2011 and 2014/2015. Data were classified based on the tests requested; as either panel (LFT or urea and electrolytes) or individual (alanine transaminase or creatinine) tests.Results: Expenditure on panel tests reduced from R340 million in 2010/2011 to R140m by 2014/2015 (reduction of R204m) and individual test expenditure increased from R34m to R76m (twofold increase). A significant reduction in LFT panel expenditure was noted; reducing from R322m in 2010/2011 to R130m in 2014/2015 (60% reduction).Conclusion: Changes in toxicity monitoring guidelines and the re-engineering of the NHLS request form successfully reduced expenditure on panel tests relative to individual tests. The introduction of order entry systems could further reduce unnecessary laboratory expenditure

Estimating implementation and operational costs of an integrated tiered CD4 service including laboratory and point of care testing in a remote health district in South Africa.

BACKGROUND: An integrated tiered service delivery model (ITSDM) has been proposed to provide 'full-coverage' of CD4 services throughout South Africa. Five tiers are described, defined by testing volumes and number of referring health-facilities. These include: (1) Tier-1/decentralized point-of-care service (POC) in a single site; Tier-2/POC-hub servicing processing < 30-40 samples from 8-10 health-clinics; Tier-3/Community laboratories servicing ∼ 50 health-clinics, processing < 150 samples/day; high-volume centralized laboratories (Tier-4 and Tier-5) processing < 300 or > 600 samples/day and serving > 100 or > 200 health-clinics, respectively. The objective of this study was to establish costs of existing and ITSDM-tiers 1, 2 and 3 in a remote, under-serviced district in South Africa. METHODS: Historical health-facility workload volumes from the Pixley-ka-Seme district, and the total volumes of CD4 tests performed by the adjacent district referral CD4 laboratories, linked to locations of all referring clinics and related laboratory-to-result turn-around time (LTR-TAT) data, were extracted from the NHLS Corporate-Data-Warehouse for the period April-2012 to March-2013. Tiers were costed separately (as a cost-per-result) including equipment, staffing, reagents and test consumable costs. A one-way sensitivity analyses provided for changes in reagent price, test volumes and personnel time. RESULTS: The lowest cost-per-result was noted for the existing laboratory-based Tiers- 4 and 5 ($6.24 and $5.37 respectively), but with related increased LTR-TAT of > 24-48 hours. Full service coverage with TAT < 6-hours could be achieved with placement of twenty-seven Tier-1/POC or eight Tier-2/POC-hubs, at a cost-per-result of $32.32 and $15.88 respectively. A single district Tier-3 laboratory also ensured 'full service coverage' and < 24 hour LTR-TAT for the district at $7.42 per-test. CONCLUSION: Implementing a single Tier-3/community laboratory to extend and improve delivery of services in Pixley-ka-Seme, with an estimated local ∼ 12-24-hour LTR-TAT, is ∼ $2 more than existing referred services per-test, but 2-4 fold cheaper than implementing eight Tier-2/POC-hubs or providing twenty-seven Tier-1/POCT CD4 services.

An integrated tiered service delivery model (ITSDM) based on local CD4 testing demands can improve turn-around times and save costs whilst ensuring accessible and scalable CD4 services across a national programme.

BACKGROUND: The South African National Health Laboratory Service (NHLS) responded to HIV treatment initiatives with two-tiered CD4 laboratory services in 2004. Increasing programmatic burden, as more patients access anti-retroviral therapy (ART), has demanded extending CD4 services to meet increasing clinical needs. The aim of this study was to review existing services and develop a service-model that integrated laboratory-based and point-of-care testing (POCT), to extend national coverage, improve local turn-around/(TAT) and contain programmatic costs. METHODS: NHLS Corporate Data Warehouse CD4 data, from 60-70 laboratories and 4756 referring health facilities was reviewed for referral laboratory workload, respective referring facility volumes and related TAT, from 2009-2012. RESULTS: An integrated tiered service delivery model (ITSDM) is proposed. Tier-1/POCT delivers CD4 testing at single health-clinics providing ART in hard-to-reach areas (<5 samples/day). Laboratory-based testing is extended with Tier-2/POC-Hubs (processing ≤ 30-40 CD4 samples/day), consolidating POCT across 8-10 health-clinics with other HIV-related testing and Tier-3/'community' laboratories, serving ≤ 40 health-clinics, processing ≤ 150 samples/day. Existing Tier-4/'regional' laboratories serve ≤ 100 facilities and process <350 samples/day; Tier-5 are high-volume 'metro'/centralized laboratories (>350-1500 tests/day, serving ≥ 200 health-clinics). Tier-6 provides national support for standardisation, harmonization and quality across the organization. CONCLUSION: The ITSDM offers improved local TAT by extending CD4 services into rural/remote areas with new Tier-3 or Tier-2/POC-Hub services installed in existing community laboratories, most with developed infrastructure. The advantage of lower laboratory CD4 costs and use of existing infrastructure enables subsidization of delivery of more expensive POC services, into hard-to-reach districts without reasonable access to a local CD4 laboratory. Full ITSDM implementation across 5 service tiers (as opposed to widespread implementation of POC testing to extend service) can facilitate sustainable 'full service coverage' across South Africa, and save>than R125 million in HIV/AIDS programmatic costs. ITSDM hierarchical parental-support also assures laboratory/POC management, equipment maintenance, quality control and on-going training between tiers.

Performance of the PointCare NOW system for CD4 counting in HIV patients based on five independent evaluations.

INTRODUCTION: Point-of-care (POC) CD4 testing can improve access to treatment by enabling decentralization and reducing patient loss-to-follow-up. As new POC CD4 technologies become available, their performance should be assessed before widespread deployment. This study reports the findings of five independent evaluations of the PointCare NOW CD4 system. MATERIALS/METHODS: Evaluations were conducted in Southern Africa (Mozambique, South Africa) and North America (Canada, USA). 492 blood samples (55 from HIV-negative blood donors and 437 from HIV-infected patients, including 20 children aged between 12 and 59 months) were tested with both the PointCare NOW and reference flow cytometry instruments. Assessment of bias, precision and levels of clinical misclassification for absolute and percent CD4 count was conducted. RESULTS: PointCare NOW significantly overestimated CD4 absolute counts with a mean relative bias of +35.0%. Bias was greater in samples with CD4 counts below ≤ 350 cells/µl (+51.3%) than in the CD4 >350 cells/µl stratum (15.1%). Bias in CD4% had a similar trend with an overall relative mean bias of +25.6% and a larger bias for low CD4 stratum (+40.2%) than the higher CD4 stratum (+5.8%). Relative bias for CD4% in children was -6.8%. In terms of repeatability, PointCare NOW had a coefficient of variation of 11%. Using a threshold of 350 cells/µl, only 47% of patients who qualified for antiretroviral therapy with reference CD4 testing, would have been eligible for treatment with PointCare NOW test results. This was 39% using a 200 cells/µl threshold. Agreement with infant samples was higher, with 90% qualifying at a 25% eligibility threshold. CONCLUSION: The performance of the PointCare NOW instrument for absolute and percent CD4 enumeration was inadequate for HIV clinical management in adults. In children, the small sample size was not large enough to draw a conclusion. This study also highlights the importance of independent evaluation of new diagnostic technology platforms before deployment.

Decentralised CD38 activation monitoring: aspects of practical implementation and standardisation.

BACKGROUND: In light of the HIV pandemic, significant strides have been made in improving treatment options for patients. Technologies to monitor the progress of a patient on such treatment have therefore also been scaled up. Immune activation as measured by CD38 mean fluorescence intensity (MFI) on CD8 T cells has been successfully shown in a clinical trial to predict response to antiretroviral therapy (ART) and reported as a cost effective real time test to supplement more costly VL testing. In this study we report transfer of this technology from the research into the routine environment. METHODS: This study was conducted in 2 parts: Firstly, fresh random samples (n=75) were tested at four time intervals (0, 24, 36 and 48 h) post-venesection to review reproducibility of CD38 MFI expression. Secondly, the CD38 MFI assay was introduced into a pilot regional testing facility and random samples (n=40) were validated against values obtained on matched samples tested at the reference laboratory. RESULTS: The CD38 assay showed acceptable accuracy and reproducibility up to 36 h (98% similarity) after venesection with some reduction in CD38 MFI to 94% at 48 h (bias<0.2MFI, %CV<5). Implementation at the secondary testing site was successful with 98% similarity (% SIM CV<5%) compared to the reference laboratory. CONCLUSION: The assay proved stable over time and could be tested until 48 h after venesection with no loss of CD38 MFI. Off-site implementation also proved successful, as such, the CD38 assay offers a reliable real time supplementary test to long-term VL monitoring of HIV infected patients on the national ART programme.

Performance evaluation of the Pima™ point-of-care CD4 analyser using capillary blood sampling in field tests in South Africa.

BACKGROUND: Point-of-care CD4 testing can provide immediate CD4 reporting at HIV-testing sites. This study evaluated performance of capillary blood sampling using the point-of-care Pima™ CD4 device in representative primary health care clinics doing HIV testing. METHODS: Prior to testing, prescribed capillary-sampling and instrument training was undertaken by suppliers across all sites. Matching venous EDTA samples were drawn throughout for comparison to laboratory predicate methodology (PLG/CD4). In Phase I, Pima™ cartridges were pipette-filled with EDTA venous blood in the laboratory (N = 100). In Phase II (N = 77), Pima™ CD4 with capillary sampling was performed by a single operator in a hospital-based antenatal clinic. During subsequent field testing, Pima™ CD4 with capillary sampling was performed in primary health care clinics on HIV-positive patients by multiple attending nursing personnel in a rural clinic (Phase-IIIA, N = 96) and an inner-city clinic (Phase-IIIB, N = 139). RESULTS: Pima™ CD4 compared favourably to predicate/CD4 when cartridges were pipette-filled with venous blood (bias -17.3 ± STDev = 36.7 cells/mm3; precision-to-predicate %CV < 6%). Decreased precision of Pima™ CD4 to predicate/CD4 (varying from 17.6 to 28.8%SIM CV; mean bias = 37.9 ± STDev = 179.5 cells/mm3) was noted during field testing in the hospital antenatal clinic. In the rural clinic field-studies, unacceptable precision-to-predicate and positive bias was noted (mean 28.4%SIM CV; mean bias = +105.7 ± STDev = 225.4 cells/mm3). With additional proactive manufacturer support, reliable performance was noted in the subsequent inner-city clinic field study where acceptable precision-to-predicate (11%SIM CV) and less bias of Pima™ to predicate was shown (BA bias ~11 ± STDev = 69 cells/mm3). CONCLUSIONS: Variable precision of Pima™ to predicate CD4 across study sites was attributable to variable capillary sampling. Poor precision was noted in the outlying primary health care clinic where the system is most likely to be used. Stringent attention to capillary blood collection technique is therefore imperative if technologies like Pima™ are used with capillary sampling at the POC. Pima™ CD4 analysis with venous blood was shown to be reproducible, but testing at the point of care exposes operators to biohazard risk related to uncapping vacutainer samples and pipetting of blood, and is best placed in smaller laboratories using established principles of Good Clinical Laboratory Practice. The development of capillary sampling quality control methods that assure reliable CD4 counts at the point of care are awaited.

A model for continuous quality control incorporating sample-to-sample assessment of optical alignment, fluorescence sensitivity, and volumetric operation of flow cytometers.

BACKGROUND: Bead count rate (BCR) monitoring successfully identifies pipetting error during single platform CD4 enumeration. Despite rigorous prescribed quality control performed, preliminary data suggested that BCR outliers could also be attributed to occasional failure of flow cytometric volumetric operation. The aim of this report was to use counting beads in a model of continuous quality control (CQC) to monitor overall flow cytometric performance (laser alignment, fluorescence stability and volumetric operation). METHODS: The proposed CQC model used FlowCheck and IMMUNOTROL blood controls daily. Extended monitoring of fluidics (FPV; beads and sheath only) and sample preparation (SPV; blood, IMMUNOPREP and beads) was done daily on five flow cytometers over five consecutive days prior to testing patient samples. Sample-to-sample CQC included monitoring BCR, selected time/fluorescence histograms (Time vs. Count; Time vs. Fluorescence and Forward Scatter vs. Fluorescence) and full peak coefficient of variation (FPCV) for 2000 samples tested. RESULTS: Prescribed quality controls showed Half Peak CV values of <2% (FlowCheck) with Immunotrol within 0.5SD of the target means. Laser stability was confirmed (FPCV values <2%). However, fluidics (volumetric operation) fluctuated as indicated by a 3.2% BCR outlier rate of 2,000 samples tested (minus pipetting error) despite optimal fluidics performance verified at start-up (FPV CV < 3%). CONCLUSIONS: Sustained laser stability was confirmed with Time vs. Fluorescence histograms, but Time vs. Count histograms were insufficient to detect intermittent volumetric failure. The proposed CQC model, incorporating BCR monitoring with time/fluorescence histograms and FPCV monitoring can identify all volumetric inconsistencies in real-time.

From research tool to routine test: CD38 monitoring in HIV patients.

BACKGROUND: CD38 expression on CD8+ T lymphocytes in HIV-infected patients is monitored by flow cytometry (FCM). There is however no consensus re CD38 protocols, analyses or result reporting within/between laboratories. Internal quality control measures (QC) were established for a standardized CD38 protocol and a system proposed for reporting CD38 fluctuation in longitudinal HIV+ patient monitoring. METHODS: A single-platform (SP) CD38/CD8 protocol was "piggy-backed" onto the standardized "panleucogating" CD45/CD4+ protocol. A weekly QC was established to monitor instrument stability (FlowSET) and absolute cell count accuracy and reproducibility (stabilized blood product, Immuno-Trol). The Mean Fluorescence Intensity (MFI) of CD38 expression on CD8(+)-lymphocytes was monitored on both stabilized blood and HIV-control samples. Linearized MFI values were determined from biological controls, i.e. healthy donor monocytes and granulocytes, and tested as a method of reporting CD38 expression on selected HIV+ patients on ART. RESULTS: The CD45/CD4/CD8/CD3 method for lymphocyte enumeration compared well with the CD38 protocol (CD45/CD4/CD8/CD38) with excellent similarity (+/-100%) and precision for absolute CD4 and CD8 counts (CVs < 5%). Fluorosphere MFI- (FlowSet, FlowCount) and color compensation values were exceptionally stable over time. CD38 MFI values established on monocytes as biological control was 4.0 and <2.0 for HIV-control lymphocytes. CONCLUSIONS: Monitoring FCM with fluorosphere MFI values, color compensation, and biological controls, can ensure that CD38 analyses are technologically stable. Flow cytometry is thus the preferred method to monitor fluctuations in CD38 MFI (CD38 molecules/cell) associated with HIV-disease progression and/or response to ART and has potential for application across instruments and centers.

Large-scale affordable PanLeucogated CD4+ testing with proactive internal and external quality assessment: in support of the South African national comprehensive care, treatment and management programme for HIV and AIDS.

BACKGROUND: In order to expand the treatment of human immunodeficiency virus-1 (HIV) infected patients in Africa, millions will require cost-effective CD4 counts. Supporting laboratories therefore, need to move away from crisis management and haphazard practices to organized pathology services. The authors reviewed the performance of the simplified single platform (SP) PanLeucogated (PLG) CD4 methodology, introduced into 52 laboratories across the South African National Health Laboratory Service (SA-NHLS), with a proactive approach to training, internal quality control (IQC), and external quality assessment (EQA). METHODS: Two-color flow cytometry for SP PLG (CD4/CD45) was combined with the sample-by-sample bead-count-rate (BCR) IQC for bead pipetting. PLG + BCR was validated versus conventional predicate SP and dual-platform (DP) 4-color flow cytometric methods used in the first world-on 1181 samples from 250 HIV+ patients followed longitudinally on anti-retroviral therapy (ART). EQA (accuracy) was performed through the United Kingdom National External Quality Assessment Scheme (UK-NEQAS). Further EQA was performed across the 52 SA-NHLS SP-PLG laboratories participating on the CD4 African Regional External Quality Assessment Scheme (AFREQAS), to assess both accuracy and/precision between NHLS PLG laboratories. RESULTS: There was virtually no bias noted between SP PLG and SP predicate methods. On DP, bias and variability increased but the errors introduced were minor without affecting CD4-related clinical decisions. The simpler 2-color PLG was less expensive with additional advantages: CD4+ T-cells were discriminated from monocytes without a need for CD3-staining, and the training was faster and easier for the trainees and trainers alike. The accuracy of SP-PLG was satisfactory: all PLG results submitted to the UK NEQAS were within +/-1 Trimmed Standard Deviation (SD) of the UK NEQAS CD4 Pool Trimmed Mean. Further, on the CD4 AFREQAS, the SA-NHLS laboratories using SP-PLG + BCR showed better precision (mean %CV = 7.2%) than the CD4 methods employed in other laboratories in Africa (mean %CV = 10.7%) or on other continents (mean %CV = 12.9%). PLG + BCR accommodated high workloads, exceeding 3,000 tests/laboratory/month, with capacity for further growth around 10% per month across the SA-NHLS. CONCLUSIONS: The superior performance of PLG + BCR over other methods has been demonstrated. In resource-conscious countries, where large-scale ART is being introduced, flow-cytometry using PLG + BCR can make a significant impact-due to simpler operation, easier training, stricter quality assurance, and better cost-efficiency. These cost-effective flow methods can legitimately replace the more cumbersome predicate technology of the first world for ART monitoring whilst accommodating an ever-expanding national ART program and consequent extremely high workloads reached country-wide.

CD8/CD38 activation yields important clinical information of effective antiretroviral therapy: findings from the first year of the CIPRA-SA cohort.

BACKGROUND: The affordable, reliable, and simplified flow cytometric method on single platform with 2-color (CD45+, CD4+) Panleucogating (PLG-CD4) is used to monitor HIV+ patients on antiretroviral therapy (ART) in South Africa (SA). Viral load (VL) assays are also used to monitor response to ART but are labor-intensive with costs that, in the long run, may remain unsustainable. Cheaper and quicker alternatives to VL such as CD38 tests on CD8+ T cells may therefore play a role in securing the continuation of ART programs in high-volume resource-restricted settings. METHODS: The single-tube PLG assay (CD45/CD4) was modified to use the full capacity of flow cytometers for 4-color immunofluorescence by including two more parameters: the CD38-activation marker on CD8 T-cells (CD8/CD38). This was introduced at baseline prior to the start of ART and then the changes of CD38+ mean fluorescent intensity (MFI) on CD8+ T-cells were then regularly assessed during follow-up-in comparison with the VL. RESULTS: A total of 103 patients received ART. By the end of their 4-, 8-, 12-, and 24-week observation periods, 29 (32.2%), 58 (64.4%), 74 (82.2%), and 83 (92.2%) had undetectable VL. This was also reflected by the gradual decrease of CD38 MFI expression on CD8+ T cells, irrespective of whether patients were "high," "moderate," or "low" CD38 responders at baseline. As expected, the CD4+ T cell counts substantially increased during the first 4 weeks from baseline 186 +/- 8.3 (SEM) to 267 +/- 12.3 cells/microl blood. But the recovery was slower over the rest of the 1-year follow-up to reach 334 +/- 18.2 cells/microl at week 48. As these CD4 increments were meager, the longitudinal follow-up of the continuously decreasing CD38 MFI values has become a particularly useful laboratory parameter to ascertain that the patients had indeed been responding well to ART. This monitoring protocol, in uneventful cases, may assist in reducing the frequency of VL testing. Conversely, a rise of CD38 MFI, if significantly higher than that seen at the previous visit even without fully reverting back to high baseline CD38-MFI values, provides an immediate indication for VL testing to judge whether or not the rebound of immune-activation is due to HIV VL-related irregularities. Therefore the CD8/CD38 test can provide the early, albeit not fully HIV-specific, warning signs about nonadherence to ART and/or developing drug resistance. CONCLUSION: This single-tube 4-color PLG-CD4 + CD8/38 activation assay (CD4/CD45/CD8/CD38), can replace the conventional 4-color CD4 protocols by substituting the redundant CD3 reagent with the informative CD38 antibody. This modification carries virtually no extra costs, while adding extra value to CD4 monitoring and enabling real-time, practical management of patients on ART. As a result, the numbers of VL tests required in patients on ART can be reduced-to save costs across our national treatment program which is already equipped with the necessary flow-cytometric screening capacity.