Point-of-care testing for HIV and TB integration of services

A thesis submitted to the Faculty of Health Sciences, university of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the Degree of Doctor of Philosophy Johannesburg, 2015The United Nations Programme on HIV/AIDS (UNAIDS) have recently released challenging new Human Immunodeficiency Virus (HIV) treatment targets to be achieved globally by 2020; all of which require concentrated efforts in scaling up laboratory testing capacity for HIV diagnosis, treatment initiation and treatment monitoring. The Global Tuberculosis (TB) Strategy have also put forth a list of ambitious goals which include reducing the number of deaths due to TB by 95% and the number of new TB cases by 90%. In South Africa, which has the highest national prevalence of HIV described globally and ranks fifth in the world in terms of TB incident cases, further integration of HIV and TB services will be needed to achieve these targets. A major challenge to successful integration of these programs however, will be the ability to diagnose and monitor the progress of both infections, a process that in South Africa, is hampered by lack of access to laboratory testing. Although public pathology laboratory service providers, such as the National Health Laboratory Service (NHLS), are responding to increasing testing demands by scaling up centralised laboratory capacity, limitations such as the need for expertise, infrastructure, space, cold-chain, maintenance, logistics and cost, are challenging full implementation and scale up. Many international organisations believe that one of the ways to successfully achieve the global HIV ‘90-90-90’ and TB targets, will be through the development and scaling up of innovative, simpler and more affordable technology approaches such as Point-of-Care testing (POCT), a view shared by the South African National Department of Health (NDoH). POCT refers to testing that is performed near or at the site of the patient with the result leading to a possible or immediate change in patient management or outcome and holds promise as a strategy to extend laboratory testing capacity. Prior to large-scale POC implementation efforts can begin, defining the difficulties and potential solutions which are likely to arise, particularly in high disease burden clinical settings need to be addressed. The main objective of this study was to investigate the feasibility, performance and operational considerations of multidisciplinary POCT in South Africa, including the development of a best practice framework to guide implementation efforts. This was achieved by performing a clinical needs assessment and engaging with government, evaluating POC technologies for HIV and TB diagnosis and/or monitoring and developing a framework for how to implement POCT in the field including quality, site and training requirements. The operational requirements for healthcare workers to perform multiple POCT in the South African clinical setting, was also determined. The assays required were based on the South African National Treatment guidelines in the period of review (2011-2014). In July 2013, the South African NDoH called a meeting with various stakeholders to provide the context for POCT in South Africa and strong emphasis was placed on HIV and TB and how POCT could expand on existing laboratory infrastructure for these diseases. Outcomes from this meeting prompted a thorough literature review on the challenges likely to be faced by large-scale POC implementation efforts. One of the key issues highlighted was the lack of evaluation data on numerous HIV and TB POC technologies available and/or in the pipeline. Even though viral load (VL) testing has been available in South Africa since 2004, the global treatment guidelines (World Health Organization) now recommend a VL test for HIV antiretroviral treatment (ART) monitoring and there are talks around the possibilities of a ‘test and treat’ strategy. In light of this, two potential POC plasma-based VL technologies available at the time were evaluated in the laboratory. The Liat™ HIV-1 Plasma Quant (IQuum Inc, MA, USA; now Roche Molecular, Branchburg, MJ, USA) and the Xpert® HIV-1 VL (Cepheid, Sunnyvale, CA) assays both demonstrated good performance and were proven to be interchangeable with existing in-country high-throughput VL laboratory platforms. Both however, require centrifugation to obtain the plasma sample and thus may be more suited to a district level facility as opposed to a ‘true’ POC environment. In light of these operational challenges, two further blood-based POC VL platforms were also evaluated, the Liat™ HIV-1 Blood Quant VL assay (IQuum, Inc) and the Alere™ q HIV-1/2 assay (Alere Technologies GmbH, Jena, Germany). Both assays identified more patients as treatment failures at the 1000 copies/ml treatment failure threshold (WHO and South African treatment guideline recommended threshold) compared to plasma VL, due to their total nucleic acid extraction protocols. Thus, if either were implemented at POC, one could expect a significant upward misclassification, increasing the number of HIV-positive patients requiring follow up VL testing and programmatic costs. Application therefore, could be niched VL testing; utilising a blood-based POC VL assay in maternity wards to diagnose HIV in new-borns; plasmabased POCT for mothers to reduce risk of transmission. POCT may not be the only solution to increasing access to laboratory testing services, and thus alternative strategies for improving access were also investigated. Dried blood spots (DBS) and PrimeStore media (a sample transport media; Longhorn Vaccines and Diagnostics, San Antonio, TX, USA) were shown to be as valuable as plasma VL for detecting HIV-positive patients failing ART at the 1000 copies/ml threshold and both solve logistical issues around sample transport and maintaining sample integrity for centralized testing. For TB diagnosis, the Xpert® MTB/RIF assay (Cepheid, Sunnyvale, CA) was evaluated to determine its appropriate placement within the South African setting. Although Xpert® MTB/RIF proved superior in performance to smear microscopy, it was originally modelled as too costly for POC placement in South Africa and was implemented into smear microscopy centres nationally. Subsequently, the complexity of the analyser maintenance and power issues has reinforced the original decision. Further potential POC TB technologies are in the development pipeline, but only one other was available for evaluation, namely the EasyNAT® detection kit (Ustar Biotechnologies, Hangzou, China). Initial laboratory evaluation results look promising but the technology is still a long way from clinical evaluation due to its laborious procedure. A further challenge identified for POCT is the lack of documented implementation science to ensure quality-assured multi-disciplinary POCT in the field. To address this, three key components of a quality testing framework were developed to ensure best practice for POCT; a clinic site readiness assessment tool, a POC training module and a quality monitoring program. The clinic site assessment checklist was developed to determine site readiness for POC placement. The POC training module included standard operating procedures, quick reference and workflow charts and a practical training component which was developed specifically with the non-laboratory trained user in mind. Both these components have been adopted and modified for use by the NHLS National Priority Program (NPP). Certain POC assays already have External Quality Assessment (EQA) material, while others had to be developed. For quality management of HIV VL technologies, a standardized plasma panel was developed to ensure molecular VL platforms are ‘fit-forpurpose’ (verification, a requirement of the laboratory accreditation process). This panel, termed SAVQA, is being manufactured and supplied to aid POC assay developers in assessing their product for the South African market, and will also be further developed for use by healthcare workers at POC. Due to the hurdles encountered with the biosafety regulations for transporting TB external quality assessment (EQA) material, a quality assessment program using dried culture spots (DCS) was also developed for TB diagnostic technologies consisting of two components; a verification and an EQA program. The DCS technology has become a global product and as of 2015 is being supplied to 20 different countries. DCS were successfully shown to be suitable for use at POC by non-laboratory trained staff. The versatility of the material has been confirmed by its expansion to other molecular TB diagnostic tests, most notably the Hain Genotype MTBDrplus assay for TB drug susceptibility testing (Hain LifeScience GmbH, Nehren, Germany). This work has been acknowledged through the Research and Development team involved in the development of the DCS program, winning three awards: the NHLS Top Award for Innovation 2013, the Gauteng Accelerator Program (GAP) Biosciences Award in 2014 and a special Social Impact award for Africa Innovations held in Morocco in 2015. Incorporating the quality components developed above, a clinical evaluation of nurse operated multidisciplinary POCT was performed. Although multiple POCT could be performed as accurately as laboratory testing on venepuncture specimens, it required dedicated staff and dramatically increased POC staff duties. It was further shown that multiple POCT could be accurately performed by a nurse on a single finger slice in order to obtain adequate blood volume to perform up to four POC tests, and that finger stick VL testing was also feasible by nurses at POC. Patients were also more willing to have up to three finger sticks performed than to have a single venepuncture specimen taken. The process of using finger sticks was further ratified by demonstrating that a single finger stick can provide up to 150μl of blood, which is sufficient to perform an array of POC tests. In spite of the feasibility of nurse based POCT, limitations of current technologies using finger stick were also realised, such as the performance of the Liat™ Quant blood assay which generated increased VL misclassification at the 1000 copies/ml treatment failure threshold (70% misclassification). This would impact programmatic costs, but this technology may have value as a diagnostic tool in key populations. The work described shows that multi-disciplinary POCT within a South African setting is achievable with appropriate clinic infrastructure, dedicated staff, training and stringent quality monitoring measures in place. The HIV and TB POC technologies evaluated were found to be as accurate as laboratory-based testing however, few meet the criteria of a ‘true’ POC device and thus further research and development is required. Based on South Africa’s testing needs, a tiered hybrid model which expands on centralized laboratory capacity through incorporating POCT into very remote, hard-to-reach areas and innovations around linkage to care efforts, may help meet ’90-90-90’ targets but will require costing/modelling and future assessments of the impact and outcome of the intervention. Much of this work presented contributed towards the development of a draft National POCT policy document in support of the national strategic plan for POCT for the management of HIV and TB in South Africa

A retrospective study characterizing the complete s open reading frame of hepatitis B virus from black children with membranous nephropathy treated with interferon alpha-2b

ABSTRACT In sub-Saharan Africa a causal relationship has been established between hepatitis B virus (HBV) infection and membranous nephropathy (MN), especially in Black children. The most common method of treatment is interferon therapy, which is however, only effective in 30-40% of patients. The reason for this is unclear. The objective of this pilot study was to determine whether mutations in the complete surface gene of HBV isolated from Black children with HBV-associated MN before, during and after treatment with interferon, had any effect on treatment response and vice versa. HBV DNA was extracted from the serum of a responder, reverter and non-responder patient before, during (4 and 16 weeks) and after (40 weeks) IFN treatment. The preS1/preS2/S region was amplified and cloned, and the clones sequenced. Sequence analyses revealed the preS2 region to be the most variable in the reverter and non-responder and HBsAg was the most variable in the non-responder. Phylogenetic analysis showed that the viral population dynamics between the responder strains and the reverter/non-responder strains differed as a result of various mutations found within the surface gene. Thus the presence of mutations in preS2 and HBsAg of the non-responding patients may carry predictive markers for nonresponse but further investigation would be needed to conclusively prove this

South Africa’s experience with provision of quality HIV diagnostic services

No abstract available

Pollen DNA barcoding:Current applications and future prospects.

Identification of the species origin of pollen has many applications, including assessment of plant-pollinator networks, reconstruction of ancient plant communities, product authentication, allergen monitoring, and forensics. Such applications, however, have previously been limited by microscopy-based identification of pollen, which is slow, has low taxonomic resolution, and few expert practitioners. One alternative is pollen DNA barcoding, which could overcome these issues. Recent studies demonstrate that both chloroplast and nuclear barcoding markers can be amplified from pollen. These recent validations of pollen metabarcoding indicate that now is the time for researchers in various fields to consider applying these methods to their research programs. In this paper, we review the nascent field of pollen DNA barcoding and discuss potential new applications of this technology, highlighting existing limitations and future research developments that will improve its utility in a wide range of applications.publishersversionPeer reviewe

Human immunodeficiency virus (HIV)-infected patients accept finger stick blood collection for point-of-care CD4 testing

INTRODUCTION HIV-infected patients require antiretroviral treatment for life. To improve access to care, CD4 enumeration and viral load tests have been redesigned to be used as point-of-care techniques using finger-stick blood. Accurate CD4 counting in capillary blood requires a free flowing blood drop that is achieved by blade incision. The aim of this study was to assess the attitude of the patients toward blade-based finger-stick blood donation. METHODS Four hundred and ninety-nine patients were included (299 patients from South Africa and 200 from Belgium). They completed a questionnaire to express their preference for finger stick or venipuncture, after undergoing both. The South African patient cohort was divided in two groups, receiving either single or multiple finger stick for CD4 and other HIV-related tests. The Belgian patients received a single finger stick for CD4 testing, and were asked to respond directly and again after two days. RESULTS The majority of the patients preferred the finger stick to the venipuncture. The perceived pain using the blade was superior to a small needle, but similar to a large needle. They preferred up to three finger sticks over one venipuncture. Up to 30% of the patients changed their mind over two days. The main reason for choosing a finger stick was continued bleeding after venipuncture. The most cited objection to finger stick was pain/soreness. CONCLUSION Patient perceptions support the implementation of donating capillary blood with bladebased finger stick during CD4 point-of-care testing.S1 File. Questionnaire for group 1 in South Africa. Patients with single finger stick. (PDF)S2 File. Questionnaire for group 2 in South Africa. Patients with multiple finger stick. (PDF)S3 File. First questionnaire in Antwerp (English). For preference immediately after finger stick. (PDF)S4 File. First questionnaire in Antwerp (Dutch). For preference immediately after finger stick. (PDF)S5 File. First questionnaire in Antwerp (French). For preference immediately after finger stick. (PDF)S6 File. Second questionnaire in Antwerp (English). For preference two days after finger stick. (PDF)S7 File. Second questionnaire in Antwerp (Dutch). For preference two days after finger stick. (PDF)S8 File. Second questionnaire in Antwerp (French). For preference two days after finger stick. (PDF)The study in South Africa was funded by Grand Challenges Canada [Grant number 0007-02-01-01, url http://www.grandchallenges.ca]http://www.plosone.orghttp://www.grandchallenges.caam2016Haematolog

The role of connected diagnostics in strengthening regional, national and continental African disease surveillance

No abstract available

Diagnosing childhood pulmonary tuberculosis using a single sputum specimen on Xpert MTB/RIF at point of care

Background. The GeneXpertMTB/RIF (Cepheid, USA) (Xpert) has proved successful for pulmonary tuberculosis (TB) diagnosis on decontaminated/concentrated induced sputum specimens from children. Capacity to perform induction in many settings is limited.Objective. To assess: (i) volumes of ‘routinely obtained’ sputum in a district-level academic hospital; (ii) whether sputum specimens not meeting Xpert-required testing volumes could still be tested; and (iii) performance of Xpert on a single paediatric sputum specimen at point of care (POC).Methods. Two sputa were collected from paediatric TB suspects (£14 years) at Rahima Moosa Mother and Child Hospital, Johannesburg, South Africa. One specimen was weighed at POC; if the volume was ≥0.1 mL but <0.5 mL, it was increased to 0.5 mL using saline. On-site Xpert testing (G3 cartridge) was performed by a dedicated laboratory technician. The second specimen was referred for TB smear microscopy and culture as per standard of care (SOC). Results. A total of 484 patients presumed to have TB (median age 24 months) were eligible for this study, performed between June 2011 and May 2012. Xpert could not be used on 4.1% of specimens because of volumes <0.1 mL, and 62.8% required addition of saline prior to Xpert testing. Xpert generated a 2.2% error and 3.7% invalid rate, compared with the SOC that rejected 2.3% because of insufficient volume and 2.3% that were contaminated. The diagnostic performance compared with culture was 62.5% (95% confidence interval (CI) 24.7 - 91) and 99.1% (95% CI 97.4 - 99.8) sensitivity and specificity, respectively, for Xpert (n=345) and 33.3% (7.9 - 69.9) and 99.5% (98.1 - 99.9) sensitivity and specificity, respectively, for smear microscopy (n=374).Conclusions. Up to 67% of ‘routinely obtained’ sputum specimens from children (£14 years) are below the required volume for Xpert testing but can be ‘topped up’ with saline. XpertMTB/RIF performed better than microscopy and generated clinically relevant, timeous results, but sensitivity did not reach the same levels as culture in children

An investigation of fingerstick blood collection for point-of-care HIV-1 viral load monitoring in South Africa

Background. Viral load (VL) quantification is an important tool in determining newly developed drug resistance or problems with adherence to antiretroviral therapy (ART) in HIV-positive patients. VL monitoring is becoming the standard of care in many resource-limited settings. Testing in resource-limited settings may require sampling by fingerstick because of general shortages of skilled phlebotomists and the expense of venepuncture supplies and problems with their distribution.Objective. To assess the feasibility and ease of collecting 150 µL capillary blood needed for the use of a novel collection device following a classic fingerstick puncture.Methods. Patients were recruited by the study nurse upon arrival for routine ART monitoring at the Themba Lethu Clinic in Johannesburg, South Africa. Each step of the fingerstick and blood collection protocol was observed, and their completion or omission was recorded.Results. One hundred and three patients consented to the study, of whom three were excluded owing to the presence of callouses. From a total of 100 patients who consented and were enrolled, 98% of collection attempts were successful and 86% of participants required only one fingerstick to successfully collect 150 µL capillary blood. Study nurse adherence to the fingerstick protocol revealed omissions in several steps that may lower the success rate of capillary blood collection and reduce the performance of a subsequent VL assay.Conclusion. The findings of this study support the feasibility of collecting 150 µL of capillary blood via fingerstick for point-of-care HIV-1 VL testing in a resource-limited setting

Comparison of Xpert MTB/RIF with Other Nucleic Acid Technologies for Diagnosing Pulmonary Tuberculosis in a High HIV Prevalence Setting: A Prospective Study

In this prospective, real-world cohort study nested within a national screening program for tuberculosis, Lesley Scott and colleagues compare the performance of Xpert MTB/RIF on a single sputum sample with different TB sputum detection technologies

Options to Expand HIV Viral Load Testing in South Africa: Evaluation of the GeneXpert® HIV-1 Viral Load Assay.

Expansion of HIV viral load (VL) testing services are required to meet increased targets for monitoring patients on antiretroviral treatment. South Africa currently tests >4million VLs per annum in 16 highly centralised, automated high-throughput laboratories. The Xpert HIV-1 VL assay (Cepheid) was evaluated against in-country predicates, the Roche Cobas Taqmanv2 and Abbott HIV-1RT, to investigate options for expanding VL testing using GeneXpert's random access, polyvalent capabilities and already established footprint in South Africa with the Xpert MTB/RIF assay (207 sites). Additionally, the performance of Xpert HIV-1VL on alternative, off-label specimen types, Dried Blood Spots (DBS) and whole blood, was investigated.Precision, accuracy (agreement) and clinical misclassification (1000cp/ml) of Xpert HIV-1VL plasma was compared to Taqmanv2 (n = 155) and Abbott HIV-1 RT (n = 145). Misclassification of Xpert HIV-1VL was further tested on DBS (n = 145) and whole blood (n = 147).Xpert HIV-1VL demonstrated 100% concordance with predicate platforms on a standardised frozen, plasma panel (n = 42) and low overall percentage similarity CV of 1.5% and 0.9% compared to Taqmanv2 and Abbott HIV-1 RT, respectively. On paired plasma clinical specimens, Xpert HIV-1VL had low bias (SD 0.32-0.37logcp/ml) and 3% misclassification at the 1000cp/ml threshold compared to Taqmanv2 (fresh) and Abbott HIV-1 RT (frozen), respectively. Xpert HIV-1VL on whole blood and DBS increased misclassification (upward) by up to 14% with increased invalid rate. All specimen testing was easy to perform and compatible with concurrent Xpert MTB/RIF Tuberculosis testing on the same instrument.The Xpert HIV-1VL on plasma can be used interchangeably with existing predicate platforms in South Africa. Whole blood and DBS testing requires further investigation, but polyvalency of the GeneXpert offers a solution to extending VL testing services