Image: Lumora Ltd.

Recent updates to HIV treatment guidelines have revealed a trend toward earlier treatment and highlighted the longer life expectancy being seen in the HIV population.^1-3 Collectively, these two factors mean that patients are receiving antiretroviral therapy (ART) for longer than ever before. In the developing world, efforts to scale up treatment of HIV have led to increases in the proportion of patients receiving combination ART (cART).⁴ However, in areas where resources are limited and conditions are challenging, finding effective ways to ensure that this treatment is and continues to be effective remains an often unattainable goal.
Recent results from the HPTN 052 trial, conducted by the HIV Prevention Trials Network on more than 1,700 sero-discordant couples (in which one partner is HIV-positive and one is not) in Africa, Asia, Latin America, and the United States, have shown that adherence to an effective ART regimen can reduce the risk of disease transmission by 96%.⁵ The ability to monitor responses to treatments and detect early resistance is even more important not only in treating individuals but also in reducing the spread of HIV.
The initiation of ART is most often determined by CD4 T-cell counts and the presence of HIV-related symptoms.⁶ However, once therapy has started, reductions in viral load become the primary indicator of therapeutic efficacy, with the aim of maintaining a viral load that is undetectable. This is because CD4 counts and symptoms are less sensitive to the success or failure of therapy.⁶ In addition, changes in CD4 cell counts are subject to the individual nature of the immunological response to ART, which has been demonstrated in resource-limited settings, restricting the utility of CD4 cell counts in predicting virological failure.⁷ Assessing viral load is accepted as the most accurate way to monitor response to treatment and detect early resistance or the need for further testing. On a larger scale, population-level viral load testing can evaluate the effectiveness of HIV treatment programs and identify regions or centers where additional support is needed.⁶
In the developed economies, recent advances in currently available technologies have provided high-volume, fully integrated, and automated systems that have revolutionized laboratory testing. However, practicalities in terms of sample

Figure 1. The BART diagnostic process. (Click image to enlarge.)

handling, contamination risk, temperature control, subjective data interpretation, and cost implications for resource-limited settings mean that tried and tested technologies from advanced economies do not always work well in the developing world. Such regions are of course where HIV infection is most prevalent and where detection and evaluation technologies are needed most. As a result, the World Health Organization is actively encouraging the development of simple, low-cost, but highly effective assays that can monitor HIV infection in resource-limited settings.⁸
This article reviews the currently available technologies for suitability in resource-limited settings and discusses a new technology that could address the significant and so far unmet needs in the developing world for HIV viral load monitoring.

Assessing HIV Viral Load in Low-Resource Settings

The following three generic methods are currently available in developing economies to detect and quantify HIV RNA: reverse transcription polymerase chain reaction (RT-PCR), nucleic acid sequence–based amplification (NASBA), and branched chain DNA (bDNA). Each method requires three steps: sample preparation and viral nucleic acid extraction, amplification of the target nucleic acid sequence or signal generated from the detection of target viral RNA, and detection and/or quantification of the amplified products.⁶
RT-PCR uses a reverse transcriptase enzyme to convert viral RNA into complementary DNA (cDNA), which then replicates and thus can be detected. In this method, RT-PCR quantifies HIV RNA, enabling determination of viral load.⁶
NASBA is an isothermal method of amplification in which target RNA is amplified by a three-enzyme system and quantified by competitive co-amplification of controls. This method removes the need for heat-stable enzymes and thermocycling instruments that are used for RT-PCR.⁶
 

Figure 2a. Raw BART output from a 10-fold dilution series of a nucleic acid target. (Click image to enlarge.)

Currently, the following four commercially available viral load assays all use of one of these general methods: Cobas AmpliPrep and Cobas TaqMan v2.0 by Roche Molecular Systems, RealTime HIV-1 by Abbott Laboratories, Versant HIV-1 RNA 1.0 assay (kPCR) by Siemens Healthcare Diagnostics, and NucliSens EasyQ HIV-1 v2.0 by bioMérieux.^4,6
Cobas TaqMan HIV-1. The Cobas TaqMan HIV-1 assay is an in vitro nucleic acid

Figure 2b. Quantification of a nucleic acid target using BART. (Click image to enlarge.)

amplification test for the quantitation of HIV-1 RNA in human plasma using the Cobas AmpliPrep Instrument for automated specimen processing and the Cobas TaqMan Analyzer or Cobas TaqMan 48 Analyzer for automated amplification and detection. It is a fully automated, real-time PCR system that targets both the gag and LTR regions of the HIV genome, which results in a lower limit of detection.⁶
The Cobas TaqMan HIV-1 assay requires only some technical skill, has a wide dynamic range, is fully automated (including sample transfer), and requires only a single room for analysis, but it has some disadvantages. In order to perform the Cobas TaqMan HIV-1 assay, some dedicated laboratory space and equipment are required, and moderate technical support is essential. The equipment is also expensive, usually beyond the reach of any developing-world facilities that can provide space and support.⁶
RealTime HIV-1. The Abbott RealTime HIV-1 assay uses real-time PCR, but unlike TaqMan, it targets the pol integrase region of the HIV genome. HIV RNA quantification is an automated process that uses the m2000sp for sample preparation and nucleic acid extraction, and the m2000rt for amplification and detection.⁶
Key advantages of the Abbott RealTime assay include its high throughput, large dynamic range, and the fact that it can be a fully automated, closed system during PCR. Additionally, the equipment can be used to assess other pathogens besides HIV. However, as with the Cobas TaqMan, this system requires dedicated equipment and space, good technical support, and a considerable financial investment.⁶
Versant HIV-1 RNA 1.0. The Versant HIV-1 RNA 1.0 assay (bDNA) is an automated assay that also targets the pol integrase region of the HIV genome, again using the sample preparation module and amplification/detection module.⁶
The Versant assay also has high throughput and can be fully automated, lowering the risk of contamination compared with

Table I. Main characteristics of the current commercial real-time HIV viral load assays. (Click image to enlarge.)

PCR. No special lab set up or separate extraction area is needed, and the equipment can also be used for assessing other pathogens. However, the main disadvantages of the Versant assay include the need for skilled technicians, good technical support, and financial resources.⁶
NucliSens EasyQ. The NucliSens EasyQ HIV-1 v2.0 uses NASBA to target the gag region for amplification and quantification of HIV RNA (see Table I). This assay couples NASBA with real-time detection by using molecular beacons that utilize the NucliSens EasyQ analyzer.⁹
The NucliSens EasyQ assay offers the advantage of high throughput with only medium skill required. It is also a fully automated, closed system. However, it requires dedicated space and equipment, and there is a risk of contamination in high-volume laboratories. Technical support is required, adding to the already high costs associated with this assay.⁶

Challenges of Viral Load Assays in Resource-Limited Settings

The currently available technologies display good sensitivity, specificity, and reproducibility, and have been optimized in terms of time to obtain results and the risk of contamination. However, variability remains between them in terms of performance, and all have limitations to their use in low-resource settings. The most significant limitation is cost. These newer technologies are several orders of magnitude more expensive compared with more traditional methods. Moreover, practicalities such as access to and cost of consumables, maintenance of suitable environmental

BART point-of-care molecular diagnostics system.

conditions, and access to a reliable electricity source prevent the application of these assays in the developing world.⁴
These technologies also require the following: invasive sample collection (venipuncture), relatively large sample sizes (200–1000 µL), complex sample preparation and testing procedures, refrigeration (2–8°C), main electricity supply, multiple components of costly equipment ($30,000–60,000), high amount of consumables per test, advanced training, technical support, and several hours to deliver results (at a cost of up to $100 per result).⁷
For an HIV-1 viral load assay to be ideally suited to resource-limited settings, different specifications are required: finger or heelstick lancets for sample collection, capability to test samples as small as 100–200 µL, minimal consumables and cost per test, heat-stable reagents, minimal and inexpensive handheld equipment that can run on batteries, minimum technician time, rapid time-to-result (ideally less than 1 hour each), and minimal training.⁷
Attempts have been made to simplify molecular diagnostic approaches in low-resource settings by using some form of endpoint visual inspection of amplification reactions. The reported benefits of such approaches are that they remove the need for more complicated detection hardware.¹⁰ However, such methods still require a means to incubate an amplification reaction at the required temperature, rely on subjective visual inspection, can introduce contamination risks by excessive handling of consumables, and fail to leave an electronic copy of the data used to derive the test result.  Furthermore, such methods can make the consumable costs much higher, especially where an additional lateral-flow methodology is employed.
Lumora has introduced the Bioluminescent Assay in Real-Time (BART), a platform for performing molecular diagnostics that allows real-time closed-tube quantitative detection of amplification by using a hardware system that can generate and store objective test results.

Overview of BART

BART is a bioluminescent reporter system for molecular diagnostics that can reduce instrument costs and open up new applications for diagnostics and disease monitoring in resource-poor settings. Requiring only a single-temperature heating block and a photo-diode light detection system, BART is designed for use with isothermal nucleic acid amplification technologies (iNAAT). It combines simple and robust chemistry and technology in real-time, closed-tube analysis (requiring minimal electrical input and temperature regulation), and less-demanding sample preparation. BART has advantages compared with several currently available systems and other systems in late-stage development, and could improve monitoring of viral load in HIV patients in the developing world.
 

BART point-of-care molecular diagnostics system.

The iNAATs generally used in molecular diagnostics produce amplicons at an exponential rate. As a consequence, pyrophosphate (PPi), a by-product of amplicon production, is also generated exponentially. By linking the exponential generation of PPi to adenosine triphosphate (ATP) synthesis, it is possible to use thermostable versions of firefly luciferase to report on the extent of amplification quantitatively via the light emitted from the luciferase (see Figure 1). Because the BART technology follows the rate of change of processes rather than making absolute measurements of concentrations, it is robust to different sample types and tolerant of large amounts of deoxyadenosine triphosphate (dATP, also a substrate for firefly luciferases) and considerable amounts of contaminating ATP and PPi.¹¹
The use of thermostable luciferases is essential to the BART technology, since most iNAATs operate at between 50° and 65°C. To date, BART has been integrated with several isothermal iNAATs based on using strand displacement polymerases such as Bst DNA polymerase.¹¹ The inherent specificity of certain methods is so high that it is sufficient to follow PPi production to infer specific amplification of a target analyte.
The light output from the BART reporter system is very different to that seen in fluorescent methods in that there is first a large increase in light emission as amplification proceeds, followed by a rapid decrease when the PPi concentration rises so far as to become inhibitory to the luciferase reaction (see Figure 1). This unusual nature of the BART outputs makes it possible to determine when a result has gone to completion without the need for complex or highly sensitive light detectors. The time taken to reach this peak of light emission is inversely proportional to the amount of target nucleic acid in the sample. Hence, BART acts quantitatively, and the time-to-result has been shown to be similar to that of fast PCR systems (see Figure 2a and 2b).¹²

Advantages of BART

BART tests can be followed in real time, allowing quantitative analysis, and are performed in a closed-vessel format, thereby reducing the risk of contamination. The unique nature of the BART biochemistry means that results can be detected using a simple light detection system (e.g., a photodiode), facilitating early detection and decision-making with no need for an expensive, highly sensitive light detection apparatus (see Figure 3). The BART technology has been shown to be more tolerant of less processed samples, due to the fact that the DNA polymerase used in iNAATs is less affected by common inhibitors. Similarly, it is possible to analyze samples that either have inherent fluorescence or are turbid, which is an issue for other techniques.
These properties enable simpler protocols to be established for a particular test as a whole. For example, where magnetic particles have been used for specific capture of DNA, it is not necessary to elute DNA from the beads before attempting amplification as with PCR. Rather, the beads can be added directly to the BART reagent, which can tolerate the presence of

Table II. Specifications of the HIV viral load BART system. (Click image to enlarge.)

magnetic beads. This enhanced sample tolerance means that sample preparation can be simplified, an advantage in resource-limited settings and challenging physical environments (see Table II).
BART is well suited to high-throughput applications, making it equally useful in both highly decentralized settings and centralized laboratories requiring high-throughput technologies. The hardware is also portable and powered by mains or a battery, culminating in a low-cost unit with a small footprint that can be used in challenging environments, including non-laboratory settings. In essence, BART is designed to be the laptop equivalent of a laboratory-based molecular diagnostic system (see Figure 3).

Future Potential

The fact that BART is easy to use and relatively low-cost means that wider adoption of this technology could be expected in laboratories where currently available methodologies are not being used, due to either ongoing high costs or the practical limitations created by the accessibility of consumables, power, and a laboratory environment that is suitable for highly sensitive preparation and testing procedures.
Previously, unreliable and inaccurate laboratory testing has caused unnecessary expenditures in resource-limited regions, promoting the misconception that laboratory testing is of little or no benefit in these settings.¹³ BART offers a simple and effective method for monitoring viral load in developing countries and could support current efforts to increase the effectiveness of and adherence to cART regimens.
Further developments of the BART technology are exploring simple array formats to allow simultaneous testing of a panel of different pathogens in a single sample of body fluid from a patient. This development would be particularly useful in a patient who has symptoms that are non-specific and there is uncertainty about the causative organism.
Larger arrays and fluid manipulation processes are also being investigated to enable digital analysis using BART. Digital PCR is a new approach to nucleic acid detection and quantification, in which the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. Samples are diluted to contain as little as a single target molecule, from which the absolute number of target molecules can be deduced using statistical analysis. From this, the starting concentration can be calculated without the need for a reference standard.
As well as the potential for quantitation of infectious organisms, digital PCR is particularly useful for identifying copy number variations and detecting rare mutations, in which high signals from wild types do not interfere. However, the complex nature of precise thermocycling, fluorescent excitation, and detection applied to large arrays means that instrumentation can be prohibitively expensive. Consequently, digital PCR has to date only been adopted for research programs. However, a digital BART technology could provide a means to simplify the required instrumentation, thereby reducing costs and making this new approach accessible for routine clinical practice in resource-poor settings.
Beyond HIV, application of a digital BART technology could provide a simple, low-cost technology in a variety of areas, including rare allele detection, cancer mutations, copy number variation, single cell research, absolute quantification of pathogens, and global food testing.

Conclusion

In the developing world, efforts to scale-up HIV treatment have not yet been matched by enhanced monitoring.⁷ As such, effective and accessible technologies that can monitor ongoing responses to HIV treatment in resource-limited settings and challenging environments are needed. While recent advances in the technologies used to monitor viral load in patients with HIV have led to the development of several new methodologies that offer enhanced sensitivity and practical application, those currently available are limited in their suitability for low-resource settings.
BART offers a new technology that facilitates the monitoring of HIV infection in resource-limited settings by overcoming obstacles such as cost, consumables, and the need for both a power source and a suitably stable environment in which to carry out complex procedures. Instead, BART is low-cost and simple to use in challenging settings and environments. As such, BART could impact the ongoing management of HIV and other infectious diseases in the developing world. Future development of a digital BART technology is expected to have wider applications across multiple areas of research, diagnostics, and monitoring, providing a low-cost and low-maintenance alternative to the more complex technologies available to date.

References

1. European AIDS Clinical Society (EACS), “HIV Treatment Guidelines; available from Internet at: http://www.europeanaidsclinicalsociety.org/guidelines.asp Accessed March 2011.
2. Department of Health and Human Services Guidelines; available from Internet at: http://www.aidsinfo.nih.gov/contentfiles/adultandadolescentgl.pdf Accessed March 2011.
3. MA Thompson, JA Aberg, P Cahn, et al., “Antiretroviral Treatment of Adult HIV Infection: 2010 Recommendations of the International AIDS Society-USA Panel,” Journal of the American Medical Association 304 (2010): 321-333.
4. C De Mendoza and V Soriano, “Update on HIV Viral-load Assays: New Technologies and Testing in Resource-limited Settings,” Future Virology 4, no. 5 (2009): 423-430.
5. HIV Prevention Trials Network (2011); available from Internet at: http://www.hptn.org/web%20documents/PressReleases/HPTN052PressReleaseFIN...
6. World Health Organization, “Technical Brief on HIV Viral Load Technologies” (2010); available from Internet at: http://www.who.int/hiv/topics/treatment/tech_brief_20100601_en.pdf  
7. A Calmy, N Ford, B Hirschel, et al., “HIV Viral Load Monitoring in Resource-Limited Regions: Optional or Necessary?” Clinical Infectious Diseases 44, no. 1 (2007): 128-134.
8. World Health Organization, “HIV Care and PMTCT in Resource-Limited Settings” (2009); available from Internet at: http://www.who.int/hiv/topics/mtct/HIV_Care_9-09.pdf
9. J Yao, Z Liu, LS Ko, et al., “Quantitative Detection of HIV-1 RNA Using NucliSens EasyQ HIV-1 Assay,” Journal of Virological Methods 129, no. 1 (2005): 40-46.
10. KA Curtis, DL Rudolph, and SM Owen, “Sequence-Specific Detection Method for Reverse Transcription, Loop-Mediated Isothermal Amplification of HIV-1,” Journal of Medical Virology 81, no. 6 (2009): 966-72.
11. OA Gandelman, VL Church, CA Moore CA, et al., “Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time,” PLoS One 5, no. 11 (2010): e14155.
12. C Smith, “Fast Real-time PCR: Getting the Information Out There” (2010); available from Internet at: http://www.biocompare.com/Articles/FeaturedArticle/1109/Fast-Real-time-P...
13. CA Petti, CR Polage, TC Quinn, et al., “Laboratory Medicine in Africa: a Barrier to Effective Health Care,” Clinical Infectious Diseases 42, no. 3 (2006): 377-382.

Laurence Tisi, PhD, is CEO and and cofounder of Lumora Ltd.

Olga Gandelman, PhD, is a senior scientist at Lumora Ltd.

Paul Weinberger, MBA, is chief marketing officer at Lumora Ltd.