Using laboratory data to assess the impact of coronavirus (COVID-19) on reflex cryptococcal antigenaemia (CrAg) testing in South Africa.

BACKGROUND: Coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first reported in Wuhan, China. Due to the rapid spread globally, it was declared a pandemic in March 2020. Social distancing and lockdown measures were introduced to limit transmission. These strategies could potentially impact the diagnosis and treatment of patients with advanced HIV who are susceptible to secondary infections like cryptococcal disease. In South Africa, reflexed cryptococcal antigenaemia (CrAg) testing and pre-emptive antifungal treatment are recommended preceding antiretroviral therapy initiation for patients with a CD4<100 cells/µl. This study aimed to assess the impact of COVID-19 on CrAg testing in South Africa. METHODS: Specimen-level data was extracted for individuals ≥15 years from the National Health Laboratory Services repository for calendar years 2018 to 2021. Test volumes and CrAg positivity were assessed at national and provincial levels, by age category and gender. The percentage change in annual and monthly CrAg test volumes for 2020 and 2021 (during lockdown levels) are compared to data reported for 2018. The monthly median CD4 and the percentage of samples with a count <25, 25-50, 51-75 and >75-<100 cells/µl were assessed. RESULTS: Specimen data of 11 944 929 CD4 results included 1 306 456 CrAg tests. Annual CD4 and CrAg test volumes declined by 22.4% and 27.8% for 2020 and 2021 respectively (relative to 2018). There were 23 670 CrAg positive outcomes in 2018 compared to 21 399 (-9.6%) and 17 847 (-24.6%) in 2020 and 2021 respectively. A monthly test volume reduction of up to 36.6%, 35.5%, 36.1% and 13.3% was reported for infection waves one to four. CrAg detection increased from 6.3% in 2018 to 7.5% in 2020. More testing was offered to males (>56%) with a higher detection rate of 8.1% in 2020. Between 81.0% and 81.8% of testing was for patients aged 20 to 49 years. The monthly percentage of specimens <25 cells/µl ranged from 30.2% (June 2019) to 35.3% (August 2020). Overall, the monthly median CD4 ranged from 39 (IQR: 15-70)(August 2020) to 45 (IQR: 19-72)(March 2019) cells/µl. In 2020, the provincial percentage change in CrAg test volumes ranged from 2.9% to -33.7%. CONCLUSION: Our findings confirmed the impact of lockdown measures on both the absolute number of CrAg tests performed and detection (increase in 2020). A smaller impact on the median CD4 was noted. The long-term impact on patient management in immune- compromised individuals needs further investigation.

Assessing CD4 rejections across a national laboratory service for 2018 in South Africa: highlighting the importance of adherence to national handbook guidelines.

The National Health Laboratory Service as the preferred pathology service provider for the public health sector in South Africa, developed a national laboratory handbook to improve the clinic-laboratory-interface. A separate primary health care laboratory handbook was developed as part of the ideal clinic initiative by the National Department of Health. This study aimed to assess adherence to these guidelines using CD4 rejections the indicator. The retrospective crosssectional study design was used to analyse national laboratory data for the period from January to December 2019. Data were analysed using SAS 9.4. Lookup tables assigned the origin (health facility/laboratory), rejection reason, and sub-reason based on the populated rejection description that was captured in the laboratory information system. The rejection rate [RR = (rejections/total volume) ´ 100] was reported at the national, provincial and district levels. There were 85,378 rejections reported for 2,844,242 tests (RR 3.0%). Data was reported for 4136 health facilities across nine provinces. The RR was higher for an origin defined as health facility (2.9%) than laboratories (0.1%). The most common rejection reason was unsuitable specimen received (RR=2.3%), representing 75% of all rejections. This rejection criteria included using the incorrect anticoagulant, clotted sample and haemolysis. The provincial RR ranged from 2.2% to 4.0%. Three districts had an elevated RR ≥6% (organisational cut-off set at RR ≤5%). This study demonstrated the value of laboratory data to assess specimen rejections and identify causes to facilitate targeted training.

Using text mining techniques to extract prostate cancer predictive information (Gleason score) from semi-structured narrative laboratory reports in the Gauteng province, South Africa.

BACKGROUND: Prostate cancer (PCa) is the leading male neoplasm in South Africa with an age-standardised incidence rate of 68.0 per 100,000 population in 2018. The Gleason score (GS) is the strongest predictive factor for PCa treatment and is embedded within semi-structured prostate biopsy narrative reports. The manual extraction of the GS is labour-intensive. The objective of our study was to explore the use of text mining techniques to automate the extraction of the GS from irregularly reported text-intensive patient reports. METHODS: We used the associated Systematized Nomenclature of Medicine clinical terms morphology and topography codes to identify prostate biopsies with a PCa diagnosis for men aged > 30 years between 2006 and 2016 in the Gauteng Province, South Africa. We developed a text mining algorithm to extract the GS from 1000 biopsy reports with a PCa diagnosis from the National Health Laboratory Service database and validated the algorithm using 1000 biopsies from the private sector. The logical steps for the algorithm were data acquisition, pre-processing, feature extraction, feature value representation, feature selection, information extraction, classification, and discovered knowledge. We evaluated the algorithm using precision, recall and F-score. The GS was manually coded by two experts for both datasets. The top five GS were reported, with the remaining scores categorised as "Other" for both datasets. The percentage of biopsies with a high-risk GS (≥ 8) was also reported. RESULTS: The first output reported an F-score of 0.99 that improved to 1.00 after the algorithm was amended (the GS reported in clinical history was ignored). For the validation dataset, an F-score of 0.99 was reported. The most commonly reported GS were 5 + 4 = 9 (17.6%), 3 + 3 = 6 (17.5%), 4 + 3 = 7 (16.4%), 3 + 4 = 7 (14.7%) and 4 + 4 = 8 (14.2%). For the validation dataset, the most commonly reported GS were: (i) 3 + 3 = 6 (37.7%), (ii) 3 + 4 = 7 (19.4%), (iii) 4 + 3 = 7 (14.9%), (iv) 4 + 4 = 8 (10.0%) and (v) 4 + 5 = 9 (7.4%). A high-risk GS was reported for 31.8% compared to 17.4% for the validation dataset. CONCLUSIONS: We demonstrated reliable extraction of information about GS from narrative text-based patient reports using an in-house developed text mining algorithm. A secondary outcome was that late presentation could be assessed.

Categorizing and Establishing CD4 Service Equivalency: Testing of Residual, Archived External Quality Assessment Scheme Sample Panels Enables Accelerated Virtual Peer Laboratory Review.

BACKGROUND: Testing of collated, curated residual archived external quality assessment (EQA) trial material, with pre-established true (consensus) values collated into 25-sample panels enables virtual peer review of a laboratory's proficiency. In this study, we introduce how archived EQAS samples/panels can establish equivalency of CD4 reporting across multiple laboratories in a national program. METHODS: Curated unused trial material from archived EQAS CD4 trials were collated into 25-sample panels comprising three sets of five-sample replicates and at least three duplicate samples. Panel-samples were tested using predicate methods of participating laboratories and proficiency determined by calculating a Standard Deviation Index (SDI) for each panel-sample reported according to retrospective consensus pooled trial outcomes. All data were plotted using MS Excel and Graphpad-Prism with SDI limits between -2 and +2 SDI to define acceptable performance. Percentage similarity analysis determined agreement. Bead-count-rate data was used to determine pipetting error. RESULTS: Tight clustering of SDI outcomes predicted acceptable laboratory proficiency with acceptable accuracy and precision as evidenced by mean SDI < 0.5 and SD of SDI < 0.5. Random pipetting error was identified with aberrant BCR. Systematic under-reading of results was noted in one lab with excellent precision but mean SDI > 1.6. Additional training requirements were evident where a respective laboratory's SD of SDI exceeded 0.7. CONCLUSIONS: Archival panel testing undertaken across a network of CD4 laboratories using the same CD4 method to simultaneously test the same panel prior to national implementation highlighted proficient laboratories and was useful for identifying sites with service deficiencies and immediate additional training needs. © 2019 International Clinical Cytometry Society.

The iron status of South African blood donors: balancing donor safety and blood demand.

BACKGROUND: Several studies in developed countries have demonstrated high levels of iron deficiency (ID) among blood donors. There is a paucity of data for developing countries where blood shortages remain a major concern. STUDY DESIGN AND METHODS: A total of 4412 donors were enrolled in the study. Specimens were collected for full blood count, iron, transferrin saturation, and ferritin assessment. Donor demographics were recorded. ID was indicated by a ferritin level of less than 20 ng/mL for men and less than 12 ng/mL for women. Anemia was defined as hemoglobin levels less than 12.5 g/dL. Regression models for predictors of ID were developed. RESULTS: A total of 17.5% of all donors had ID, with 16.3% prevalence in women and 18.6% in men. Low hemoglobin had the highest association with ID (adjusted odds ratio [AOR], 11.078; 95% confidence interval [CI], 7.915-15.505); male donors had twice the odds of ID compared to female donors (AOR, 2.501; 95% CI, 1.964-3.185), while increasing age was associated with lower odds (AOD, 0.965; 95% CI, 0.956-0.975). Among male donors, an interdonation interval of less than 3 months (AOR, 2.679; 95% CI, 1.929-3.720) was associated with ID. Compared to other females combined, colored female donors (AOR, 2.335; 95% CI, 1.310-4.160) had higher odds and black female donors (AOR, 0.559; 95% CI, 0.369-0.845) lower odds of ID. CONCLUSION: ID is common among South African donors; low hemoglobin, gender, ethnicity, and past donation history is independently associated with ID. Recommendations aimed at protecting donor health may increase blood shortages in South Africa.

Cryptococcal antigen positivity combined with the percentage of HIV-seropositive samples with CD4 counts <100 cells/µl identifies districts in South Africa with advanced burden of disease.

INTRODUCTION: Cryptococcal meningitis (CM) is an opportunistic fungal disease with a high mortality among HIV-positive patients with severe immunosuppression (CD4 count <100 cells/µl). Reflexed screening for cryptococcal antigen (CrAg) in remnant blood samples was initially piloted at selected CD4 testing laboratories of the National Health Laboratory Service (NHLS) prior to the implementation of a national screening programme using a lateral flow assay (LFA) (IMMY, Norman, OK, USA). The aim of this study was to assess CrAg positivity nationally, per province and district in combination with the percentage of CD4 samples tested with a CD4 count <100 cells/µl to identify areas with advanced HIV/CrAg disease burden. METHODS: CrAg and CD4 laboratory result data were extracted from the NHLS corporate data warehouse. Monthly test volumes were used to assess CrAg test volumes and coverage, while bubble charts were used to display the relationship between CD4 <100 cells/µl, CrAg positivity and number of positive CrAg samples by district. ArcGIS software was used to spatially report CrAg positivity. RESULTS: CrAg screening coverage was stable at around 96% after November 2016. Samples with a CD4 <100 cell/µl and CrAg positivity were also stable over the study period at 10% and ~5% respectively. The highest CrAg positivity was reported for the Kwa-Zulu Natal province (7.3%), which also had the lowest percentage of samples with a CD4 <100 cells/µl (7.2%). Uthungulu and Umkhanyakude districts had the highest CrAg positivity (9.3% and 8.9% respectively). Ethekwini and Johannesburg Metro districts contributed to 22% of the total number of CrAg-positive samples tested across South Africa for the period reported. CONCLUSION: Existing CD4 testing services were used to rapidly scale up CrAg reflex testing in South Africa. Districts with advanced HIV and CrAg disease burden were identified that need further investigation of patient management interventions.

Estimating the cost-per-result of a national reflexed Cryptococcal antigenaemia screening program: Forecasting the impact of potential HIV guideline changes and treatment goals.

INTRODUCTION: During 2016, the National Health Laboratory Service (NHLS) introduced laboratory-based reflexed Cryptococcal antigen (CrAg) screening to detect early Cryptococcal disease in immunosuppressed HIV+ patients with a confirmed CD4 count of 100 cells/µl or less. OBJECTIVE: The aim of this study was to assess cost-per-result of a national screening program across different tiers of laboratory service, with variable daily CrAg test volumes. The impact of potential ART treatment guideline and treatment target changes on CrAg volumes, platform choice and laboratory workflow are considered. METHODS: CD4 data (with counts < = 100 cells/µl) from the fiscal year 2015/16 were extracted from the NHLS Corporate Date Warehouse and used to project anticipated daily CrAg testing volumes with appropriately-matched CrAg testing platforms allocated at each of 52 NHLS CD4 laboratories. A cost-per-result was calculated for four scenarios, including the existing service status quo (Scenario-I), and three other settings (as Scenarios II-IV) which were based on information from recent antiretroviral (ART) guidelines, District Health Information System (DHIS) data and UNAIDS 90/90/90 HIV/AIDS treatment targets. Scenario-II forecast CD4 testing offered only to new ART initiates recorded at DHIS. Scenario-III projected all patients notified as HIV+, but not yet on ART (recorded at DHIS) and Scenario-IV forecast CrAg screening in 90% of estimated HIV+ patients across South Africa (also DHIS). Stata was used to assess daily CrAg volumes at the 5th, 10th, 25th, 50th, 75th, 90th and 95th percentiles across 52 CD4-laboratories. Daily volumes were used to determine technical effort/ operator staff costs (% full time equivalent) and cost-per-result for all scenarios. RESULTS: Daily volumes ranged between 3 and 64 samples for Scenario-I at the 5th and 95th percentile. Similarly, daily volumes ranges of 1-12, 2-45 and 5-100 CrAg-directed samples were noted for Scenario's II, III and IV respectively. A cut-off of 30 CrAg tests per day defined use of either LFA or EIA platform. LFA cost-per-result ranged from $8.24 to $5.44 and EIA cost-per-result between $5.58 and $4.88 across the range of test volumes. The technical effort across scenarios ranged from 3.2-27.6% depending on test volumes and platform used. CONCLUSION: The study reported the impact of programmatic testing requirements on varying CrAg test volumes that subsequently influenced choice of testing platform, laboratory workflow and cost-per-result. A novel percentiles approach is described that enables an overview of the cost-per-result across a national program. This approach facilitates cross-subsidisation of more expensive lower volume sites with cost-efficient, more centralized higher volume laboratories, mitigating against the risk of costing tests at a single site.

Analysis of HIV disease burden by calculating the percentages of patients with CD4 counts <100 cells/µL across 52 districts reveals hot spots for intensified commitment to programmatic support.

BACKGROUND: South Africa (SA)'s Comprehensive HIV and AIDS Care, Management and Treatment (CCMT) programme has reduced new HIV infections and HIV-related deaths. In spite of progress made, 11.2% of South Africans (4.02 million) were living with HIV in 2015. OBJECTIVE: The National Health Laboratory Service (NHLS) in SA performs CD4 testing in support of the CCMT programme and collates data through the NHLS Corporate Data Warehouse. The objective of this study was to assess the distribution of CD4 counts <100 cells/µL (defining severely immunosuppressed HIV-positive patients) and >500 cells/µL (as an HIV-positive 'wellness' indicator). METHODS: CD4 data were extracted for the financial years 2010/11 and 2014/15, according to the district where the test was ordered, for predefined CD4 ranges. National and provincial averages of CD4 counts <100 and >500 cells/µL were calculated. Data were analysed using Stata 12 and mapping was done with ArcGIS software, reporting percentages of CD4 counts <100 and >500 cells/µL by district. RESULTS: The national average percentage of patients with CD4 counts <100 cells/µL showed a marked decrease (by 22%) over the 5-year study period, with a concurrent increase in CD4 counts >500 cells/µL (by 57%). District-by-district analysis showed that in 2010/11, 44/52 districts had >10% of CD4 samples with counts <100 cells/µL, decreasing to only 17/52 districts by 2014/15. Overall, districts in the Western Cape and KwaZulu-Natal had the lowest percentages of CD4 counts <100 cells/µL, as well as the highest percentages of counts >500 cells/µL. In contrast, in 2014/15, the highest percentages of CD4 counts <100 cells/µL were noted in the West Rand (Gauteng), Vhembe (Limpopo) and Nelson Mandela Bay (Eastern Cape) districts, where the lowest percentages of counts >500 cells/µL were also noted. CONCLUSIONS: The percentages of CD4 counts <100 cells/µL highlighted here reveal districts with positive change suggestive of programmatic improvements, and also highlight districts requiring local interventions to achieve the UNAIDS/SA National Department of Health 90-90-90 HIV treatment goals. The study further underscores the value of using NHLS laboratory data, an underutilised national resource, to leverage laboratory test data to enable a more comprehensive understanding of programme-specific health indicators.

Establishing a cost-per-result of laboratory-based, reflex Cryptococcal antigenaemia screening (CrAg) in HIV+ patients with CD4 counts less than 100 cells/µl using a Lateral Flow Assay (LFA) at a typical busy CD4 laboratory in South Africa.

INTRODUCTION: Cryptococcal meningitis is a major cause of mortality and morbidity in countries with high HIV prevalence, primarily affecting patients whose CD4 are < = 100 cells/µl. Routine Cryptococcal Antigen (CrAg) screening is thus recommended in the South African HIV treatment guidelines for all patients with CD4 counts < = 100 cells/µl, followed by pre-emptive anti-fungal therapy where CrAg results are positive. A laboratory-based reflexed CrAg screening approach, using a Lateral Flow Assay (LFA) on remnant EDTA CD4 blood samples, was piloted at three CD4 laboratories. OBJECTIVES: This study aimed to assess the cost-per-result of laboratory-based reflexed CrAg screening at one pilot CD4 referral laboratory. METHODS: CD4 test volumes from 2014 were extracted to estimate percentage of CD4 < = 100 cells/µl. Daily average volumes were derived, assuming 12 months per/year and 21.73 working days per/month. Costing analyses were undertaken using Microsoft Excel and Stata with a provider prospective. The cost-per-result was estimated using a bottom-up method, inclusive of test kits and consumables (reagents), laboratory equipment and technical effort costs. The ZAR/$ exchange of 14.696/$1 was used, where applicable. One-way sensitivity analyses on the cost-per-result were conducted for possible error rates (3%- 8%, reductions or increases in reagent costs as well as test volumes (ranging from -60% to +60%). RESULTS: The pilot CD4 laboratory performed 267000 CD4 tests in 2014; ~ 9.3% (27500) reported CD4< = 100 cells/µl, equivalent to 106 CrAg tests performed daily. A batch of 30-tests could be performed in 1.6 hours, including preparation and analysis time. A cost-per-result of $4.28 was reported, with reagents contributing $3.11 (72.8%), while technical effort and laboratory equipment overheads contributed $1.17 (27.2%) and $0.03 (<1%) respectively. One-way sensitivity analyses including increasing or decreasing test volumes by 60% revealed a cost-per-result range of $3.84 to $6.03. CONCLUSION: A cost-per-result of $4.28 was established in a typical CD4 service laboratory to enable local budgetary cost projections and programmatic cost-effectiveness modelling. Varying reagent costs linked to currency exchange and varying test volumes in different levels of service can lead to varying cost-per-test and technical effort to manage workload, with an inverse relationship of higher costs expected at lower volumes of tests.

Performance Evaluation of the Becton Dickinson FACSPresto™ Near-Patient CD4 Instrument in a Laboratory and Typical Field Clinic Setting in South Africa.

BACKGROUND: The BD-FACSPresto™ CD4 is a new, point-of-care (POC) instrument utilising finger-stick capillary blood sampling. This study evaluated its performance against predicate CD4 testing in South Africa. METHODS: Phase-I testing: HIV+ patient samples (n = 214) were analysed on the Presto™ under ideal laboratory conditions using venous blood. During Phase-II, 135 patients were capillary-bled for CD4 testing on FACSPresto™, performed according to manufacturer instruction. Comparative statistical analyses against predicate PLG/CD4 method and industry standards were done using GraphPad Prism 6. It included Bland-Altman with 95% limits of agreement (LOA) and percentage similarity with coefficient of variation (%CV) analyses for absolute CD4 count (cells/µl) and CD4 percentage of lymphocytes (CD4%). RESULTS: In Phase-I, 179/217 samples yielded reportable results with Presto™ using venous blood filled cartridges. Compared to predicate, a mean bias of 40.4±45.8 (LOA of -49.2 to 130.2) and %similarity (%CV) of 106.1%±7.75 (7.3%) was noted for CD4 absolute counts. In Phase-2 field study, 118/135 capillary-bled Presto™ samples resulted CD4 parameters. Compared to predicate, a mean bias of 50.2±92.8 (LOA of -131.7 to 232) with %similarity (%CV) 105%±10.8 (10.3%), and 2.87±2.7 (LOA of -8.2 to 2.5) with similarity of 94.7±6.5% (6.83%) noted for absolute CD4 and CD4% respectively. No significant clinical differences were indicated for either parameter using two sampling methods. CONCLUSION: The Presto™ produced remarkable precision to predicate methods, irrespective of venous or capillary blood sampling. A consistent, clinically insignificant over-estimation (5-7%) of counts against PLG/CD4 and equivalency to FACSCount™ was noted. Further field studies are awaited to confirm longer-term use.

Observed full blood count and lymphocyte subset values in a cohort of clinically healthy South African children from a semi-informal settlement in Cape Town.

BACKGROUND: The paediatric full blood count and lymphocyte subset reference intervals used by the National Health Laboratory Service (NHLS), South Africa (SA), are taken from two international reference interval publications. Differences in reference intervals suggest that international data sets may not be appropriate for use in SA. OBJECTIVE: To study immunohaematological values of a group of clinically healthy children from an informal settlement in Cape Town, SA, to assess whether international paediatric reference intervals used by the NHLS are appropriate. METHODS: A cross-sectional study of 207 female and 174 male HIV-uninfected children living in an informal settlement in Cape Town was performed. Full blood counts, automated differential counts and lymphocyte subset analysis were done using internationally accepted technologies. Data were categorised by age and reference intervals compiled using medians and 95% confidence intervals (CIs). Gender comparisons were calculated by non-parametric tests. RESULTS: Although median and 95% CI values differed slightly, physiological trends for red cell, platelet, white blood cell differential and lymphocyte subsets were similar to international reference intervals currently in use at the NHLS. Benign ethnic neutropenia was not a significant finding, and gender-specific intervals were not necessary until 12 years of age. Lower overall median values for haemoglobin and haematocrit, and higher median values for mean cell volume and red cell distribution width, were noted. Assessment of haemoglobin, red cell distribution width and calculated Mentzer ratios suggested underlying iron deficiency in 14.2% of participants. CONCLUSION: Paediatric immunohaematological reference intervals observed in this study are similar to, and support continued use of, international paediatric reference intervals. Underlying iron and related nutritional deficiencies may be contributing to lower haemoglobin levels noted in local children. A larger nationwide study, including all ethnic groups, is recommended.

Local reference ranges for full blood count and CD4 lymphocyte count testing.

OBJECTIVE: Recent advances in full blood count and CD4 technology, coupled with the changing population demographics of the Gauteng region, have necessitated reevaluation of the reference ranges currently in use. METHODS: A cross-sectional study of 631 female and 88 male HIV-negative participants from the Gauteng region was performed. Full blood count, automated differential and CD4 count analyses were done using the latest internationally accepted technology. Reference ranges were compiled from the 2.5th and 97.5th percentiles for both male and female participant groups, and gender and ethnic comparisons calculated by non-parametric tests. RESULTS: Results of 41 females were removed from the statistical analysis because their results were suggestive of possible anaemia. Full blood count reference interval comparison confirmed gender-specific differences in red blood cell and platelet parameters. Ethnic-specific differences were found for some red blood cell parameters in the black female cohort. In addition, black males and females both generally had lower neutrophil and higher lymphocyte counts than a combined Asian/Caucasian/coloured ethnic group. CONCLUSION: Comparison of the currently calculated reference ranges with published data and reference values in use indicated that a separate ethnic-specific reference range should be introduced for the percentage/absolute neutrophil count and percentage lymphocytes. In addition, locally derived reference ranges for red cell distribution width (RDW) and CD4 percentage of lymphocytes should be implemented for routine diagnostic testing.