Human immunodeficiency virus or HIV is an RNA retrovirus that progresses into acquired immunodeficiency syndrome (AIDS) over a long period of time. The retrovirus attacks CD4+ T cells compromising the immune system in fighting infectious diseases [1]. HIV was first extracted from a patient in 1983 [2]. Since then, there are approximately 76 million individuals infected with HIV-1 worldwide. More antiretroviral treatment options are becoming available over time ^[3][4][3,4]. HIV can be classified as Type 1 or Type 2, with HIV-2 being less infectious and uncommon [5]. HIV-1 is generally prevalent in East Africa while HIV-2 is found in West Africa but epidemics exist in India, Brazil, Portugal, and Guinea-Bissau, among other countries ^[5][6][7][8][5,6,7,8]. In the United States, adolescents, African American men, and young adults (13–19 years) have higher incidences of HIV infection, according to past data ^[9][10][11][9,10,11]. Health disparities and gender play a role in HIV analysis because they are linked ^[12][13][12,13]. Removing healthcare barriers and providing primary and secondary AIDS prevention can reduce the disparity in care [14]. Additionally, the correlation between poverty and HIV incidence has shown conflicting data to determine a true linkage ^[15][16][15,16].

Advances have not only been made in treatment but also in HIV diagnostics. The detection of HIV was conventionally performed through lab-intensive examinations but has progressed to modern-day “lab on a chip” modalities. Streamlining the processes of detection offers a convenient method for on-the-go HIV recognition for early-stage disease identification [17]. The current gold standards for diagnosis and monitoring include flow cytometry, PCR, and ELISA. However, these techniques are expensive, time-consuming, and require skilled technicians to use them. The standards set by the World Health Organization (WHO) characterize point-of-care testing as affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable, or ASSURED [18]. By following the WHO’s guidelines for point-of-care testing, novel and innovative microfluidic devices can bridge the gap in testing and managing HIV in resource-limited areas. Evaluation of an HIV prognosis requires complex lab infrastructure and high cost for viral load testing, which may not be readily available in resource-limited settings [19]. This necessitates the need for faster, sensitive, and easily transportable microdevices that can ensure available testing. Advances in technology permit the production of handheld devices and microfluidics, including lensless imaging with smartphones and multiplexing [18]. Furthermore, CD4+ T cell counting, sample preparation, and nucleic acid molecular diagnostics, as a result, have been enhanced with the production of these new microfluidic devices [17]. As point-of-care devices become readily available, high-throughput methods can speed up population testing; however, this may come as a tradeoff to portability or processing time ^[18][20][18,20]. The capabilities of these tests can extend to HIV screening, load monitoring, and infant diagnosis [18]. To compete with the currently used conventional methods, such as flow cytometry, PCR, and ELISA, these devices have to be reliable and effective as well as inexpensive to be used in resource-limited settings.

2. HIV, CD4+ T Cells, and AIDs

HIV virions are approximately 100 nm in diameter and are encased in an envelope with surface glycoproteins. The interior capsid surrounds two identical single-stranded RNA strands [5]. Once the virus attaches and fuses with the CD4+ T cells, the reverse transcriptase generates viral dsDNA. Integrase fuses the HIV genome to the CD4+ T cell’s DNA-permitting host transcription. As more HIV proteins are produced and assemble into functional HIV particles, they are released from the CD4+ T cell through budding [21]. This repeated cycle causes a continuous decrease in CD4+ T cells through a caspase-1-mediated pyroptosis or caspase-3 activation ^[22][23][22,23]. Furthermore, the immune system is activated from HIV gene products Vpu, Nef, and Tat, which allow the virus to increase replication ^[24][25][24,25]. Late-state progression of HIV leads to AIDS due to the compromised immune system. Typically, AIDS is defined as less than 200 cells/µL for CD4+ T cells [26]. This increases the risk for infections to arise, due to the immunocompromising effects of HIV. Antiretroviral treatment can assist in the prevention of AIDS but monitoring CD4+ T cell count and viral load through testing and point-of-care services can help in disease management [27]. The primary tests for AIDS detection are inclusive to HIV detection, such as ELISA and viral load tests. These diagnostic tests serve to detect HIV markers, such as reverse transcriptase, gp120, or p24, or by amplifying HIV DNA to detectable levels. Alternatively, analytes can include HIV antibodies, such as anti-gp41 or anti-gp120. Currently, there is no cure for HIV and treatment options rely on suppressing viral replication with the use of highly active antiretroviral therapy (HAART) ^[28][29][28,29].

3. Sample Preparation

A major issue in POC HIV diagnostic devices is the lengthy sample preparation time that varies across various technologies and assays. Microfluidic devices for rapid HIV detection should have minimal sample preparation to be efficient and should provide rapid results. Unfortunately, current devices involve extensive sample preparation steps that prolong the overall time it takes to achieve diagnostic results. Traditional benchtop methods, such as PCR and ELISA, require expertise and extensive sample preparation but scalability to microfluidic devices offers rapid quantification of viral proteins and detectable loads to provide on-site results. Conventional off-chip sample preparation involves the preparation, mixing, washing, and buffering of reagents, which can take up to a few hours. Benchtop PCR, for example, requires the generation of cDNA and primer mixing, which alone can take 1 to 3 h. Automated detection systems are able to rapidly quantify virions and pre-load the microfluidic devices with the necessary reagents in a pre-packaged format. This has been implemented for zika virus detection, with detection times up to 40 min. The on-heating chip capability, temperature control, and reagent pre-loading provide the means to rapidly quantify viral loads in patient samples with zika virus ^[30][36]. The incorporation of these systems into HIV diagnostic tools have been shown to provide rapid results, from 60 to 90 min, while providing accurate results ^[31][32][33][37,38,39]. Novel HIV diagnostic technology, such as paper- and flexible material-based assays, provide a variety of designs with a simple fabrication process and the capability to be mass produced. Paper-based detection methods have been shown to decrease the detection time by reducing the sample preparation time and consolidating the entire assay process to take up to 60 min ^[34][35][40,41].

4. Microfluidic Devices for Point-of-Care Applications

Microfluidic devices are small-scale chip-based devices that contain miniaturized channels and chambers to facilitate chemical reactions. Physical forces, such as electrokinetics and capillary action, are able to mix the samples on-chip. Often times, low volumes of reagents in the microliter range are used for detection ^[36][43]. Miniaturizing laboratory techniques into an on-chip reaction can be a tedious process that places limitations on material fabrication and costs, reagent volumes, and scalability of reactions ^[37][44]. Therefore, novel methods to fabricate affordable microfluidic devices are imperative because they can bypass the inherent limitations in providing devices that can rapidly assess HIV status with great accuracy. Unfortunately, not all chip materials or fabrication processes are streamlined or cost-effective.  In this section, we describe Tthe challenges, limitations, and advantages of various diagnostic assays with a focus on affordable microfluidic devices utilized for HIV detection in resource-limited settings are described belows.

4.1. Polymerase Chain Reaction (PCR)-Based Devices

PCR is a technique that requires a thermocycler due to each stage requiring different temperatures. This method exponentially increases the nucleic acid’s quantity in each cycle. This is represented as 2ⁿ, where n is the number of cycles. The three stages are denaturing, annealing, and extending. Denaturing separates the DNA strands on which primers attach during the annealing stage. The strands are extended during the final stage, amplifying the DNA. The requirement of a thermocycler provides a hurdle for resource-limited areas because it is expensive and requires a trained technician. Another method, known as quantitative PCR (also known as real-time PCR), utilizes fluorophores to produce real-time readings. The optical filters used are expensive and offer a bottleneck in cost for resource-limited areas ^[38][45]. Reverse transcription PCR (RT-PCR) and qPCR can be used to measure gene expression by synthesizing cDNA from mRNA. In the case of HIV, viral mRNA can be detected by these machines for analysis of viral load testing.

4.2. Isothermal Amplification-Based Devices

4.2.1. Loop-Mediated Isothermal Amplification Devices

Loop-mediated isothermal amplification (LAMP) is a molecular test for nucleic acid amplification as an inexpensive substitute for PCR. Compared to the temperature fluctuations in PCR, LAMP utilizes a constant temperature between 60 and 70 °C. LAMP uses 4 to 6 primers that are extended by DNA polymerase for molecular amplification, with a similar principle to PCR ^[39][55]. However, LAMP requires no thermal cycling and is a highly specific, fast, and portable diagnostic test for infectious diseases ^[40][41][56,57]. Recently, RT-LAMP has been extensively used for COVID-19 testing and provides applications in HIV detection as well ^[42][43][58,59]. This favorable nucleic acid amplification test (NAAT) is preferred over PCR for field use and is capable of real-time detection and measuring fluorescent intensity ^[44][60]. Electricity-free RT-LAMP devices have been previously constructed, powered by an alternative source of energy. In order to overcome the hurdle of electricity availability in resource-limited countries, Singleton et al. devised an electricity-free non-instrumented nucleic acid amplification (NINA) device paired with a nucleic acid lateral flow (NALF) platform. The heating system involves an exothermic reaction from magnesium oxidation, which transfers heat to the phase change material, palmitic acid. Biplex detection of HIV and β-actin, an internal control, was performed from normal human plasma; however, the inclusion of β-actin primers decreased HIV-1 detection sensitivity. Despite this, HIV detection was reliable and performed under 80 min with the NINA-paired NALF device for RT-LAMP ^[45][61]. 

4.2.2. Recombinase Polymerase Amplification

Recombinase polymerase amplification (RPA) assay is a form of isothermal amplification. It uses fewer primers compared to LAMP and can be combined with a fluorescent probe ^[46][68]. The underlying mechanism behind RPA involves the use of a recombinase protein to bind primers, forming a complex. This complex scans for homologous sequences in DNA and then the primers are inserted by the recombinase. Afterwards, the recombinase is deconstructed, and DNA polymerase is able to elongate the primers ^[47][69]. Similar to LAMP, no thermal cycler is required, which makes this method reliable over PCR for portability, speed, and cost-effectiveness ^[48][70]. There is minimal sample preparation and RPA is rapid due to exponential DNA replication, which can produce results within 20 min for HIV and other viruses ^[49][50][51][71,72,73]. RPA can be used with reverse transcriptase, known as RT-RPA, to detect RNA molecules by synthesizing the corresponding cDNA ^[47][69]. Additionally, RPA is cost-effective compared to PCR as costs can go as low as $4.45 ^[52][74].

4.3. ELISA

Enzyme-linked immunosorbent assays (ELISA) or enzyme immunoassays (EIA) operates on the principle of antigen or antibody binding to identify molecular interactions. Antibodies are proteins produced by immune cells, such as plasma cells, in response to an infection. Pathogens express antigens on their surface, which can bind to an antibody, resulting in the formation of an antigen–antibody complex. These antigen–antibody complexes can elicit the termination of pathogens through neutralization, agglutination, precipitation, or opsonization (complement fixation). ELISA has varying methodologies depending on the method used. Direct, indirect, sandwich, and competitive ELISA all use antibodies to bind to antigens but vary in the order of binding. The substrates used are most commonly horse radish peroxidase or alkaline phosphatase because they generate a color change in the assay ^[53][80]. Early detection of HIV can be completed with ELISA with a follow-up Western Blot for a confirmatory diagnosis ^[54][81]. An indirect ELISA is normally conducted for HIV detection from a blood or saliva sample and has a greater sensitivity than direct ELISA ^[53][80]. HIV antigen p24 can be detected through ELISA during the onset of a symptomatic primary infection ^[55][82]. The currently used tests are fourth-generation assays, which are highly sensitive and specific with 100% sensitivity and 99.5% specificity ^[56][57][58][83,84,85]. Fourth-generation assays are able to detect the p24 antigen while reducing the amounts of present false negatives and false positives. Developing a point-of-care diagnostic device with ELISA can permit highly sensitive and specific detection of diseases in resource-limited settings. However, a major drawback of ELISA testing involves a long wait time, often 6 to 8 h, to obtain results ^[53][80]. Various microfluidic ELISA devices have been made with variations, such as the implementation of glass capillaries or removing an enzyme label ^[59][60][86,87]. One ELISA innovation is known as the mChip assay, which was tested in Rwanda. The mChip is cost-effective, portable, and can diagnose both HIV and syphilis. Comparable to benchtop methods, the HIV detection sensitivity and specificity are 100% and 96%, respectively, with this device ^[61][88].

4.4. ELISA Alternatives

While benchtop methods, such as PCR and ELISA, are considered gold standards for molecular detection and protein quantification, some new microfluidic devices have been put to the test ^[62][63][64][104,105,106]. A novel device known as hierarchical nanofluidic molecular enrichment system (HOLMES) consists of a series of microchannels between different stages. The first stage has the microchannels stacked vertically but as the stage progresses, the number of microchannels decreases. The final stage has one microchannel. This device operates through electro-osmosis and gravitational flow. Biomolecules are concentrated at the first stage but are then transferred to the second stage and then reconcentrated. This process repeats until the biomolecules reach the final stage, improving the concentration performance ^[65][107]. This method is capable of detecting nucleic acid and proteins at low concentrations in human sera. HOLMES is capable of detecting HIV p24 proteins with concentrations as low as 10 aM within 60 min ^[63][105]. ELISA has a detection limit around 1 pM with a longer time to obtain data, from within hours to a day ^[66][108]. Another highly sensitive automated device implemented by Hughes and Herr, which is capable of multiplexing, can detect HIV within 60 min. Known as µWestern blot, it is a rapid quantitative assay of a traditional Western blot but miniaturized for microfluidic use ^[67][109].

5. Smartphone-Based Devices

Smartphones and cellular devices are commonly used devices throughout the world, both in developed and developing countries. Smartphones are user-friendly and most provide an interactable touch interface. Most individuals in developed countries have access to or own a smartphone, as indicated by 81% of Americans having ownership of smartphones in 2019 ^[68][118]. Even in developing countries, an increasing number of individuals have access to a smartphone. According to a 2017 survey, 51% of individuals in South Africa have access to a smartphone, while 91% have access to a cellular device. Additionally, smartphone ownership has been increasing over the years, indicating greater access to cellular devices in rural or underserved areas ^[69][119]. With the multivalent capabilities of smartphones, they can be used in point-of-care settings for disease detection. Some systems have been developed using smartphones for microscopy, colorimetry analysis, and genetic testing ^{[70][71][72][73]}[92,120,121,122]. By incorporating smartphones into HIV detection, improved patient monitoring, epidemiological tracking, and early-stage diagnoses can be facilitated. Current smartphone-based devices have been used to diagnose and detect infectious diseases, including HIV ^{[70][74][75][76][77]}[92,123,124,125,126]. One study performed RT-LAMP with HIV-1 and used a smartphone for fluorescence imaging for viral load interpretation (Figure 5). The incorporation of a smartphone replaces the need for detection equipment, such as a fluorescence microscope ^[78][115]. This is cost-effective as this equipment is expensive and may not be readily available in resource-limited areas ^[48][79][70,127]. Furthermore, traditional laboratory assays, such as ELISA, can be developed on a chip and then imaged through a smartphone. Chen et al. created a platform which interfaces with a mobile device to conduct ELISA ^[80][116]. The smartphone triggers the device to supply energy to a printed circuit board that can hold the microfluidic chip for ELISA. Imaging can be conducted through the smartphone and then transmitted to a computer for processing or analysis can be conducted on the smartphone with programmable applications ^[80][116]. A study published in 2017 used microelectromechanical piezoelectric surface acoustic wave (SAW) sensors to diagnose HIV with smartphones. This highly sensitive proof-of-concept study detected HIV within seconds by speeding up the diagnostic process through the use of smartphones. This method is equipment-free, is affordable by cutting out expensive equipment, and reduces the risk of false positives by multiplexing arrays of biochips. The sensing area is composed of functionalized quartz used to capture the targeted proteins, such as p24. The limit of detection and lowest detected concentration associated with this device are 1.1 nM and 2 nM for anti-p24 antibodies, respectively. Furthermore, the speed at which results are delivered are very rapid because HIV antibody concentrations can be interpreted in 10 s after sample insertion. The limitations associated with this study include manual functionalization, which can cause variability from biochip to biochip, and manual pipetting for sample insertion, which poses a risk for contamination ^[81][114]. Innovative devices using smartphones, such as this one, can meet the WHO’s ASSURED criteria for point-of-care services in underserved areas. In another experiment, Gray et al. used SAW biosensors with a smartphone-connected prototype reader for a digital readout for HIV detection from clinical samples. The samples were electronically interpreted very rapidly, with results provided in under a minute. The dual-channel biochips used were functionalized for HIV proteins gp41 and p24 detection. Functionalization was conducted by inkjet printing, which kept variability between biochips low. Once tested, the assay demonstrated 100% sensitivity for anti-gp41 detection and 100% overall specificity. However, the anti-p21 biomarker sensitivity was 66.1% ^[82][128]. A caveat associated with smartphone-based technology is related to cybersecurity. Storing sensitive medical data on a smartphone application can lead to privacy and security concerns ^[83][117].

6. Conclusions

The implementation of point-of-care devices for HIV diagnostics in resource-limited settings can serve to bridge the need for increased testing and a lack of resources. Development of new technologies, including smartphones, offers a wide modality of assays that can be used for disease detection. Additionally, paper and flexible assays provide an avenue for inexpensive materials to be used for assays, which minimizes the cost of purchasing or developing expensive equipment. Traditional laboratory methods are resource-intensive and some require trained personal to interpret the results compared to point-of-care devices. Miniaturizing benchtop machines translates their functionality to a microscale for rapid analysis. Microfluidic point-of-care devices are also highly sensitive and specific, similar to their benchtop counterparts. They are also inexpensive due to requiring low-cost materials for fabrication and can be reusable or disposable. Rapid HIV testing can be accomplished with these various technologies but the option of which method is ideal differs based on the needs and environmental or financial restrictions. Resource-limited areas may lack complex laboratory infrastructure, which can make point-of-care devices preferable for HIV detection. Cost-effective methods stray away from PCR as thermocyclers are expensive; therefore, LAMP or RPA can be used instead because isothermal methods do not require the use of a thermocycler. Moreover, future applications of microdevices for diagnostics will head in the direction of smartphone applications. Modern smartphones have a powerful processing capability and are able to take images in a wide wavelength of light. This permits smartphones to be used as a power source for microfluidic devices or host applications that can analyze data. In resource-limited regions, emphasis should be placed on using RPA or LAMP compared to tedious methods, such as PCR or ELISA, for HIV diagnosis. Emerging technologies, such as smartphone-based detection, paper-based methods, and novel fabrication methods, should be tested in these regions to evaluate their efficacy for HIV detection. In areas that are not constrained by infrastructure or resources, PCR or ELISA should be used as the optimal standards for HIV detection. The future direction for HIV diagnosis and monitoring needs to place an emphasis on low-cost materials with rapid results and low wait times. Conventional detection methods are costly and often take a long time (hours to days) to achieve reportable results. Resource-limited and underserved regions are unable to have the proper infrastructure and equipment for accurate diagnosis and monitoring, developing the need for low-cost diagnostic systems.