Recent advances in HIV diagnostics: Point-of-care CD4⁺ T-cell count & viral load assays

Human Immunodeficiency Virus (HIV) infection continues to be a major global public health issue, claiming over 40 million lives to date1. During 2022, worldwide, ~86 per cent of the people living with HIV (PLH) became aware of their infection status, ~76 per cent were receiving antiretroviral therapy (ART) and ~71 per cent were virally suppressed. Recently, the Joint United Nations Programme on HIV/AIDS has proposed a new ambitious global target of ‘95-95-95’ by 2025, which is anticipated to have a significant influence on the quality of life of PLH. This strategy promises to ensure that 95 per cent of all PLH would be aware of their HIV infection status, 95 per cent of those aware of their HIV positive status would receive ART and 95 per cent of all individuals on ART would attain viral suppression2. Therefore, the role of laboratory diagnostics becomes pivotal in the prevention and control of HIV.

HIV/antiretroviral therapy monitoring

While a cure for HIV infection remains elusive, it has transformed into yet another manageable chronic infectious disease, enabling PLH to live long and have a quality of life with highly potent antiretroviral (ARV) drugs, which are currently in use3. However, the patients on treatment need to be constantly monitored to evaluate ART effectiveness and the potential development of drug resistance to one or more components of the ARV drug regimen.

The World Health Organization (WHO), Geneva, has developed the ‘universal test and treat’ (UTT) programme as a strategy for HIV elimination contrary to the previous ‘deferred treatment’, which was based on CD4⁺ T-cell count and/or WHO clinical staging criteria. UTT is an approach, which emphasizes that all at risk population should be screened for HIV infection and those diagnosed with HIV infection should be initiated on early ART irrespective of their CD4⁺ T-cell count and the WHO clinical staging criteria. Several countries, including India (implemented in 2017), have adopted the ‘test and treat’ policy4.

The progression of HIV disease varies across individuals, making it crucial to accurately stage the infection, predict its course and monitor its progression. Two laboratory tests, CD4⁺ T-cell count and plasma viral load (PVL) measurements are two approaches employed; the PVL measures viral replication, while the CD4⁺ T-cell count correlates with the functional implications of the immune system in the HIV-infected individual.

CD4⁺ T-cell count in monitoring

Flow cytometry-based CD4⁺ T-cell count has held substantial importance in the clinical management of HIV since the early days of HIV discovery. Apart from assessing the magnitude of immunodeficiency in infected individuals, the CD4⁺ T-cell count has played a crucial role in ascertaining the eligibility for starting ART and administering prophylactic treatment against opportunistic infections (OIs). Furthermore, it serves as a valuable tool for monitoring immune restoration in patients undergoing ART.

There was a significant demand for the development of specialized CD4⁺ T-cell count technologies and low-tech and low-cost alternatives when ART roll-out was made available on a large scale with global efforts, particularly in countries with limited economic resources and high prevalence of the disease56. During the early 2000s, a range of compact and dedicated flow cytometers for CD4⁺ T-cell count technologies were introduced, primarily for the benefit of resource-limited settings. These newer methodologies demonstrated elegant performance characteristics and provided reliable laboratory results78.

Following the release of new HIV treatment guidelines by the WHO (2016), the central role of CD4⁺ T-cell count in the clinical management of HIV infection got diminished, owing to the introduction of ‘test and treat’ and ‘rapid ART initiation’ policies9. Nonetheless, the CD4⁺ T-cell count continues to be an invaluable tool for guiding the treatment of OIs in remote clinical settings with constrained availability of PVL monitoring, particularly amongst individuals with advanced HIV disease manifestations10. Furthermore, with immunological non-responder cases being reported to be >30 per cent among PLH, the concurrent use of both PVL and CD4⁺ T-cell count is warranted for their effective clinical management1112.

In recent years, there has been notable progress in the development of simple point-of-care (POC) CD4⁺ T-cell count technologies. These new-generation technologies primarily utilize microfluidic cassettes and digital image-based analyzers, aimed at improving the availability of cost-effective monitoring for HIV-infected individuals in low- and middle-income countries (LMICs). The performance and testing reliability of these POC instruments have been well documented13. Furthermore, the turnaround time for the test result with POC CD4⁺ T-cell count assay is <40 min, resulting in a notable decrease in the number of patients who get lost to follow up.

As of now, the WHO has pre-qualified (WHO-PQ) only four POC CD4⁺ T-cell count devices, which are (i) FACSPresto Near-Patient CD4 Counter (Becton Dickinson, San Jose, USA), (ii) CyFlow Counter System with CD4 Easy Count kit (Sysmex Partec GmbH, Gorlitz, Germany), (iii) Alere PIMA CD4 Analyzer (Alere Technologies GmbH, Jena, Germany) and (iv) VISITECT CD4 Advanced Disease (Omega Diagnostics Ltd, Alva, UK)14. FACSPresto and PIMA equipment are ultra-compact, portable instruments that employ disposable test cartridges for CD4⁺ T-cell counts using both venous and finger-prick blood samples. Venous and finger-prick blood is loaded directly into the cartridge, and these smallsize instruments are designed to be used by phlebotomists or nurses with minimal training requirements. The key advantages with the finger-prick blood-based tests are; (i) being less invasive than venipuncture, (ii) use of minimal blood volume and (iii) not requiring a skilled phlebotomist. CyFlow Counter is a compact, robust and fully equipped desktop flow cytometer requiring venous whole blood to run the test. The CyFlow Counter is easier to install (AlignFree technology), and this ‘plug-and-play’ system does not need additional setup time. On the other hand, semi-quantitative CD4⁺ T-cell count assays have also been recently introduced, and one such example is Omega VISITECT CD4 Advanced Disease assay (Omega Diagnostics, Alva, UK), which does not require any instruments, and is a disposable POC assay to determine CD4⁺ T-cell count as either <200 or >200. In the current scenario of UTT, this semi-quantitative CD4⁺ T-cell count assay holds promise in offering prophylactic treatment for OIs, particularly in resource-limited settings, where the PVL often becomes inaccessible.

HIV viral load monitoring

With no available HIV cure, the main objective of administering ART is to attain HIV virological suppression. This suppression is characterized by an HIV PVL of <1000 copies/ml, as specified by the WHO. The recent WHO guidelines recommend to perform routine viral load (VL) monitoring for early diagnosis of therapeutic failure that includes a review of HIV PVL results in six and 12 months after ART commencement, and annually thereafter3. The PLH on ART who successfully attain virological suppression have enhanced immune restoration, leading to a significant reduction in morbidity and mortality15. Therefore, monitoring HIV PVL in PLHA on ART remains critical. Although numerous molecular testing platforms are available for precise and efficient measurement of HIV PVL on a large scale, these approaches still require expensive instrumentation, high-level infrastructure facilities and highly skilled laboratory personnel. Despite rapid increase in the capacity for laboratory-based (centralized) PVL testing in LMICs and efforts to make it universally available, the adoption of widespread VL monitoring faces various challenges.

Regular monitoring of PVL is crucial not only for maintaining the health of PLH but also for the public health significance it beholds. When individuals maintain an undetectable VL for at least six months, it contributes to limiting the spread of HIV from one to another (undetectable equals untransmittable or ‘U=U’). This concept is based on strong scientific evidence from four key randomized clinical trials (RCTs) with sero-discordant couples, including HIV Prevention Trials Network study protocol 052, an international multi-centric study that included Indian sites, Chennai and Pune141516.

Point-of-care plasma viral load testing

Over the past few years, a number of POC HIV PVL tests have been introduced, and there are also several emerging technologies under development. Of which, some are still in different stages of clinical validation. As of now, there are two assays1718 which have been pre-qualified by the WHO: (i) Xpert HIV-1 VL (Cepheid AB, Solna, Sweden) and (ii) m-PIMA HIV-1/2 VL (formerly Alere q HIV-1/2 Detect) (Abbott Rapid Diagnostics GmbH, Jena, Germany). The Xpert HIV-1 VL assay uses real-time reverse transcriptase–polymerase chain reaction (RT-PCR) with molecular beacons principle and amplifies HIV-1 Groups M, N and O, including recombinant forms, CRF01_AE, CRF02_AG and CRF03_AB. As an additional advantage, similar types of cartridge tests can be utilized to diagnose other infections such as sexually transmitted infections and tuberculosis. The test requires 1 ml plasma, and the lower limit of detection is 40 copies/ml. The m-PIMA equipment is a sturdy, durable, battery-powered equipment specifically designed for completely automated processing, allowing for on-site testing in field settings. The assay methodology is based on RT-PCR with competitive reporter hybridization. Apart from its capability to quantify VLs of all HIV-1 groups, including M, N and O, the m-PIMA is the sole POC assay that can also measure HIV-2 VL. However, the assay’s lower limit of detection is 800 copies/ml. This ready-to-use cartridge-based test has a capillary port to load 50 μl of plasma (from whole blood, venous or capillary).

Both Xpert and m-PIMA assays use sealed cartridges that are to be inserted into assemblies of single-module automated equipment to run the assay, and the total turnaround time to result is ~70-90 min. As the cartridge has built-in internal quality-control measures, there is no need to wait for batch sample testing. However, the disadvantage of these two WHO-PQ assays is the requirement of a refrigerated centrifuge to separate plasma from the blood samples. Studies evaluating the clinical performance of the currently available POC PVL assays against conventional standard laboratory assays in high- and low-income settings demonstrated good correlation with excellent sensitivity and specificity31920. RCTs investigated the clinical impact (viral suppression as an outcome) of POC HIV-1 PVL for PLH receiving ART in LMICs, demonstrating promising outcomes21. However, there are several other POC VL assays in clinical validation [e.g. SAMBA I PSQ (DRW Ltd, SJ, USA), SAMBA II WBSQ (DRW Ltd, SJ, USA), Roche Cobas Liat system (Roche Diagnostics, Rotkreuz, Switzerland) and TrueNat (Molbio Diagnostics, Goa)] and a few others in late stages of the development22.

Conclusions & the way forward

The currently available POC VL tests are not true POC, but rather ‘near’-POC tests, as plasma separation requires a refrigerated centrifuge. The true POC HIV VL tests should be performed using whole blood by either venipuncture or finger-prick sampling. Hence, there is a need to develop equipment-free solutions for plasma separation. The potential of POC HIV VL tests to decentralize and expand VL coverage is immense. These can significantly enhance treatment success, enable early detection of drug resistance and reduce the risk of onward HIV transmission. Therefore, the urgent need for simpler and feasible true POC HIV VL tests is evident.