Perspective For reprint orders, please contact:
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HIV diagnostics: challenges and opportunities Accurate HIV diagnostic testing continues to pose challenges, but there are also opportunities for assay performance improvements in key areas for specific intended-use settings. The genetic diversity of HIV can result in false and discordant results in assays that fail to detect new variant strains. The use of antiretroviral therapies has resulted in drug-resistant variants that require monitoring by sequencing and genotyping methods. Nucleic acid testing is the most sensitive and reliable platform for detection, but it is costly and limited to centralized testing facilities, making implementation difficult in resource-limited settings where HIV has hit the hardest. Rapid antibody tests suitable for point-of-care use are becoming more accessible in resource-limited settings, but these tests may not detect HIV during the acute infection stage. Emerging antigen/antibody combination assays are viable alternatives to nucleic acid testing for diagnosis of recent infections. Although patient monitoring (e.g., via CD4+ T-cell count and viral load determination) and infant diagnoses still rely on clinical laboratory-based testing, point-of-care options are being developed. There are other technical challenges to HIV diagnostic testing and emerging biodetection technologies that may be able to address them, but they are not yet proven. KEYWORDS: biodetection
n
diagnostic n drug resistance n HIV n rapid test
Eric Y Wong1 & Indira K Hewlett†1 Laboratory of Molecular Virology, Center for Biologics Evaluation & Research, Food & Drug Administration, 8800 Rockville Pike, Building 29B, Room 4NN16, Bethesda, MD 20892, USA † Author for correspondence: Tel.: +1 301 827 0795 Fax: +1 301 827 0998
[email protected] 1
Global burden of HIV & its management In 2007, there were estimated to be 30–36 million people living with HIV/AIDS globally and nearly 3 million incident cases, including 400,000 newly infected children under the age of 15 years (90% of whom resided in Africa) [201] . Among newborns who contracted the virus through maternal transmission, approximately half did not survive more than 2 years [1,2] . While the pandemic has stabilized in Africa since 2001, the number of reported cases increased in other regions of the world during the same period [201] . Interventions for primary prevention have generated mixed results. Circumcision is effective at preventing up to 60% of female-to-male transmissions [3–9] . Recent vaccine and microbicide candidates showed a protective effect by reducing sexual transmission rates with an efficacy of approximately 30% [10,11] , which is a stark contrast to previous vaccine and microbicide trials where candidates failed to protect against, or even enhanced, the acquisition of HIV [12–15] . An effective vaccine will probably be the most important factor in controlling the pandemic, but it may still be years before one is developed [13,15,16] . Physical barriers, such as condoms, can be up to 95% effective against transmission when used consistently, but accessibility, acceptance and adherence on a population-wide level may be difficult to achieve [17] . Preliminary results are expected
in 2010 from clinical trials investigating the effectiveness of antiretroviral drugs as pre-exposure prophylactics (PrEP) against HIV [18–20] . However, an intervention such as PrEP would require a continuous supply of product, educational programs on proper use and a committed effort to adhere on the part of the patient [18,21] . Antiretroviral therapy (ART) significantly suppresses HIV viral loads in infected individuals, greatly reduces HIV-related mortality and transmission rates, and enables healthcare providers to treat the disease as a manageable chronic condition [22] . Taken daily as a life-long treatment or over a controlled 28‑day regimen as a postexposure prophylactic by those who experience a high-risk event such as a sexual encounter with an infected individual or a needle-prick exposure by a healthcare worker, antiretroviral drugs have been responsible for the remarkable declines in postinfection morbidity and mortality over the last 20 years [22,23] . Due to dramatic effect of ART, ‘test and treat’ methods have been adopted as a potentially important strategy in the fight against HIV [24] . In the test and treat approach, a premium is placed on universal testing, followed by placing individuals who test positive for HIV immediately on ART to minimize their infectiousness to others [3,5] . Modeling predicts that this strategy could reduce HIV incidence and mortality to less than 1 case per 1000 people within 10 years of implementation [25] .
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Perspective | Wong & Hewlett However, accurate point-of-care (PoC) diagnostic and monitoring tests (in support of treatment) that are suitable for resource-limited settings (RLS) and also widely accessible antiretroviral drugs will be needed for this to be possible. Nucleic acid testing (NAT) is the most accurate and reliable screening platform for HIV detection and is widely used in blood banks for donor testing [26] . Diagnostic platforms, such as ELISAs, are in routine use for the detection of anti-HIV IgG/IgM antibodies, and the newer ELISAs simultaneously detect antibodies and the HIV viral capsid protein, p24 antigen (these are known as Ab/Ag combo or ‘fourth generation’ assays) [27] . However, NAT and ELISA platforms are limited to hospitals, clinics and laboratories, and are common only in developed countries owing to the cost and infrastructure requirements. Moreover, NAT assays and ELISAs are suboptimal in RLS because many of those in need of testing live in remote locations with limited access to clinics. The use of portable, rapid tests for decentralized PoC testing in remote areas and shipment of dried-blood spot (DBS) samples collected on filter paper to clinics for testing have been effective in diagnosing HIV infection, however [28,29] . Diagnostics for RLS have not received nearly as much support as drug and vaccine programs because medicines are less expensive, but the recognition of HIV (and other infectious pathogens) as a major public-health burden has bolstered resources for the research and development of diagnostic tools [30,31] . There are opportunities for diagnostics to play a significant role in HIV management, but there are also many challenges to be overcome, as will be discussed below (Table 1) . Rapid & PoC testing in RLS In developed countries, most patients infected with HIV have access to health programs (providers, medicines, diagnostic and monitoring tests) that support treatment. However, this model of specialist physician care paired with laboratory monitoring is not operationally feasible for those who live in RLS, and a decentralized approach toward HIV testing is more effective in these areas [32] . Diagnostic tools for these settings should meet a stringent set of criteria to be of practical use. Tests should be accurate, inexpensive, rapid, simple to administer and robust. Moreover, it would be ideal if a test could function without electricity, refrigerated reagents or complex instrumentation as these may not be available [33] . 400
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Antibody-detecting membrane immunochromatographic assays provide an excellent compromise between accuracy, cost, rapidity and overall effectiveness for PoC use in RLS [34] . Briefly, there are two major platforms; flow through and lateral flow rapid tests. In the flow through format, filter paper (porous membrane) is treated with proteins that target anti-HIV antibodies. A blood sample (i.e., whole blood, plasma or serum) is applied to the filter and any anti-HIV antibodies present will be immobilized on the filter. Following a wash, the addition of a gold nanoparticle (NP) label that binds to the antibodies generates a visible color change to reveal the test result [35] . Lateral flow systems are more prevalent, involve only a single step and are similar to a home pregnancy test. A blood sample added to the device migrates across a filter by a carrier buffer and encounters several distinct regions on the filter [35] . It first contacts a conjugate zone, which contains an anti-HIV IgG/IgM capture antibody conjugated to a gold NP that freely migrates with the carrier. Next is a capture zone with antigen immobilized to the membrane and any anti-HIV antibody present in the original sample will now be attached to the gold NP conjugate and bound by the antigen. The formation of this sandwich results in a visible color change [36,37] . These rapid tests generate a result within 30 min and are portable, which minimizes loss to follow-up as the outcome is immediately available and increases accessibility for decentralized HIV testing in remote areas. There are challenges to implementing effective, widespread PoC testing with rapid formats. Quality assurance programs should be in place to evaluate rapid tests before they are put to use, monitor test performance in given geographic regions and develop appropriate algorithms with clear instructions regarding how to proceed with confirmatory testing in the event of an initial positive test result [38–40] . In addition, the personnel employing these tests in the field must be properly trained not only in the technical aspects of the assay, since errors in operation or improper interpretation will result in an inaccurate diagnosis, but also to adequately counsel the patient following a diagnosis. Although current rapid tests are almost exclusively based on the detection of anti-HIV antibodies, they have median sensitivities and specificities that exceed 98–99%, even when used in field evaluations in Africa [34] . future science group
HIV diagnostics: challenges & opportunities
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Table 1. Challenges facing current HIV diagnostics. Desired HIV diagnostic capability
Current challenges
Accuracy (high sensitivity and specificity)
Assays require specific capture reagents; unreliability due to HIV diversity; emerging strains hard to detect Most NAT assays and ELISAs are multistep and require scanners; rapid formats require confirmatory testing The cost of consumables and instrumentation for NAT assays and ELISAs is high, as is procuring tests for population-wide testing Current assays are prone to error; panels of representative specimens are required for calibration; algorithms required to standardize testing NAT assays and ELISAs are laboratory based; equipment is required for SAPP and analysis Rapid ultrasensitive tests that detect nucleic acid or antigen in whole blood are needed Most NAT assays and ELISAs are multistep and require scanners; rapid formats must follow specified algorithms Conventional rapid tests are suboptimal; frequent monitoring may be required Need enhanced detection labels (nanoparticles); sensitive nonlabel platforms not yet widely available; assays should target nucleic acid or antigen; background noise Genotyping assays rely on accurate sequence databases and may not detect low-frequency strains; phenotyping assays are too costly and complex Patients must visit or submit samples to clinics with instrumentation and trained personnel Standard assays are designed to detect previously discovered strains; ultra-deep sequencing technologies are currently too costly May require device compartmentalization or microarrays; assay design complex; may be PCR-dependent; increases cost Lack of multiplex assays; detection procedures unreliable for coinfections Conventional tests cannot differentiate between vaccine- and virus-induced antibodies Long turnaround time; more rapid and sensitive assays needed Requires automation to minimize human error; multistep; complex; assay dependent
Rapid detection (~minutes) Low cost (per data point or device) Incidence testing Portability (functionality at the point-of-care or bedside) Diagnosing acute infection Simplicity (test can be conducted by the untrained) Infant testing (up to 18 months of age) Low level of detection Drug-resistance testing Treatment monitoring (CD4 + T-cell count and viral load) Detecting emerging strains Multiplexing (simultaneous detection of multiple pathogens or handles multiple assay formats) Detecting coinfections in resource-limited settings Testing of vaccine recipients Testing for organ and tissue transplantation Consistent sample acquisition, preparation and processing
NAT: Nucleic acid testing; SAPP: Sample acquisition, preparation and processing.
The newest ELISAs are Ab/Ag combo assays designed to detect both antibody and antigen simultaneously [27] . Compared with assays that detect antibodies alone, Ab/Ag combo assays are more accurate (detecting nearly 90% of infected individuals who had been missed by an initial antibody screening test), able to reduce the diagnostic window period (the time lag between exposure and actual detectable levels of HIV by a diagnostic assay) by almost 7 days because p24 is detectable prior to seroconversion, and may minimize false-negative results in patients carrying strains that vary from endemic circulating forms, since p24 is well conserved [27] . The development of Ab/Ag combo rapid tests will create opportunities for accurate testing in RLS [38] . The ability to collect DBS samples, prepared by depositing drops of blood from a finger or heel prick onto filter paper, has resolved some of the difficulties associated with storage and transport of specimens from areas where there is no future science group
field testing. When they arrive at the clinic or laboratory, the filter paper samples are placed in a lysis buffer to release the targets of interest (i.e., antibody, antigen or nucleic acid) into solution for analysis. DBS testing has proven to be effective in diagnosing HIV [28,41–43] , but drawbacks of this approach include reduced sample recovery owing to nonspecific binding to the membrane (reducing sensitivity), possible decomposition of critical biomarker molecules after long storage times (reducing sensitivity) [29] , and potential losses to follow-up because a diagnosis is not given to the patient at the time of the visit. Efforts aimed at building up local health programs and infrastructures would expand access to DBS testing services. Infant & acute infection testing Infants cannot be tested accurately using antibody-detecting assays until they are 18 months of age because the presence of maternal www.futuremedicine.com
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Perspective | Wong & Hewlett antibodies could mask their true HIV status [28] . The current standard of care is to start the newborn on ART while performing DNA NAT at 2 and 4 weeks of age, with two negative test results being a confirmation of no transmission [44] . Frequent monitoring would be needed for breastfeeding infants since transmission can occur through breast milk [45] . Standard practices in RLS include observing the onset of clinical symptoms as indicators of HIV status and preparing DBS to be transported to a laboratory for NAT or an ELISA [28] . There are ultra-sensitive p24 assays that are almost as sensitive as NAT under certain conditions for pediatric testing and monitoring [46] ; however, similar to NAT, the reagent requirements for p24 assays may pose logistical problems. There are algorithms that describe the use of rapid tests in infants under 18 months of age based on evidence that there is a correlation between a negative rapid test result and a negative test result by molecular assays [28] , and this could be an alterative when other tests are unavailable. In adult infections, it takes approximately 3 weeks, but perhaps as long as 6 months, before seroconversion takes place to generate detectable anti-HIV antibodies [47] . Transmissibility of HIV is high during the acute stage of infection (the first 1–3 weeks of infection) when viral load levels ramp-up in the host and antibodies may not be detected, which underscores the need for reliable testing of recent infections to inform patients of their status to minimize secondary spread to contacts and partners [24] . This can be addressed with expanded access to DBS testing or Ag/Ab combo rapid tests and education programs to encourage those who experience a high-risk exposure to immediately seek testing. Estimating incident infection rates The ability to accurately distinguish incident infections that took place within the past few months from long-standing (>1 year) infections is important for monitoring HIV transmission patterns in a given population and helps guide public-health programs aimed at curbing the spread of HIV. Typical antibody-detecting assays do not differentiate between recent and long-standing infections, but samples that are confirmed to contain anti-HIV antibodies can be applied to incidence tests that are designed to detect recent infections (within 6 months of sampling). This general approach is termed the serological testing algorithm for recent HIV seroconversion (STARHS) [48] . 402
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Examples of incidence testing formats include the BED, avidity, IDE-V3 and detuned assays, and they will be briefly described. The BED, a commercial enzyme immunoassay (EIA) used by the US CDC, detects the proportion of anti-HIV IgG present in a specimen relative to the total amount of IgG, which is an indicator of disease progression and provides an estimate of the time period since seroconversion [49,50] . Avidity is a measure of the affinity between antibodies and their antigens, the strength of which increases over time as the antibodies mature [51] . Avidity assays are modified ELISAs designed to investigate the maturity of the anti-HIV antibody response by using a chaotropic agent (guanidine or urea) to disrupt low-avidity antibody–antigen interactions. The greater the decrease in assay signal relative to a control sample (to which a neutral buffer is added), the lower the avidity of the anti-HIV antibodies and the more recent the infection. The laboratory-based IDE-V3 EIA employs a series of oligopeptides derived from two conserved immunogenic regions of HIV and uses a mathematical formula that draws on the reactivity of recent and long-standing specimens to the peptides from each region to estimate the recency of infection [52,53] . Detuned assays were the first tests described for detecting incident infections and involve increasing the dilution of a known anti-HIV antibody-positive sample and reducing the incubation time to deliberately lower the sensitivity of an EIA [53] . Long-standing infections usually have higher titers of antibody and are thus differentiated from recent infections by the greater level of detuning required before a negative test result is registered. The challenge is that when incidence assays are calibrated to determine the assay cut-off signals for recent infections according to STARHS, scientists rely on panels of specimens that may not be representative of the population, have approximated seroconversion dates, and/or are taken from patients who exhibit very different immunological responses to HIV (or are on ART) [48] . Validation of the assays also depends on similar panels or mathematical models based on prevalence data that are difficult to confirm [50] . Consequently, there is always a certain degree of uncertainty associated with these assays. When the accuracy of incidence tests were systematically reviewed from published reports, sensitivity estimates ranged from 42 to 100% (89% overall median) for incident infections and specificity estimates ranged from 35 to 100% (98% overall median) for long-standing, nonincident infections [54] . future science group
HIV diagnostics: challenges & opportunities Furthermore, when assay-derived population incidence estimates were aggregated and compared with reference estimates, the differences ranged from 0 to 483% (median difference of 26%) [54] . Although these data do suggest that, at present, incidence testing may not be very accurate, especially in regions with diverse HIV populations such as Africa [55] , STARHS methodologies can produce more reliable data if stakeholders take the opportunity to work together to establish standards for calculating cut-off values, algorithms for using multiple assays in concert to improve accuracy, and a framework for comparing different assays. In addition, there is now a focus on collecting large numbers of donor specimens for the purpose of disease research [56,57] , and expanding HIV sample repositories will give scientists greater access to representative samples for use in STARHS calibration and validation protocols, which in turn should improve the performance of incidence assays. HIV diversity enables escape from detection The prevalence, incidence rates, transmission profiles and circulating recombinant forms (CRFs) of HIV vary considerably from region to region. While HIV‑1 subtype B is the predominant form found in developed regions, such as North America, western Europe, most nations of South America and Australia, subtypes A, B and C, and their recombinant forms predominate in Asia, eastern Europe, and eastern South America, and virtually every non-B subtype can be found in Africa [58] . In the west central African nation of Cameroon, CRFs are rampant as viruses readily recombine, adding to the complexity of circulating HIV strains [59] . This expanding genetic diversity raises concerns over the ability of diagnostic assays to detect unique CRFs. Antibody-detecting assays and NAT rely on the presence of specific epitopes and genetic sequences, respectively, for HIV detection. Strain variants that do not carry these specific epitopes and sequences, or present other reactive ones, will produce inaccurate assay results [60,61] . In one study, 240 plasma samples from Cameroonian individuals were tested against a panel of commercially available immunoassays and NAT assays, and the results were discordant outcomes between assays, differences in the reactivity when compared with samples taken from US patients and undetectable recombinant forms [61] . This is evidence that the prevalence of strains endemic to each part of the world should be a consideration in the design of diagnostic assays future science group
| Perspective
based on reagents (e.g., synthetic antigens or PCR primers) that are tailored to be ‘region specific’. It is also important that highly conserved markers (e.g., p24 antigen) or gene sequences are targeted in an assay. Sequence data generated from surveillance and characterization studies from around the globe will aid in the development, evaluation and validation of serological and molecular assays to help address the challenge of HIV diversity [40] . Presence of coinfections HIV compromises immune function and leaves individuals vulnerable and susceptible to opportunistic infections. Globally, four of the most devastating HIV coinfections are TB, malaria, and HCV and HBV. The worldwide prevalence of TB is approximately 2 billion people and it is one of the most common HIV coinfections and leading causes of death [3,62,63] . It is estimated that there are over 500 million cases of malarial infection annually, mostly in Africa, and dual infection of HIV and malaria facilitates the geographical expansion of each [64] . HCV and HBV infections are also highly prevalent among the HIV population [65,66] . These coinfections are not only widespread and increase mortality rates, but they also reduce the accuracy of HIV tests [62,65–69] , creating a need for diagnostics that can quickly and reliably account for these coinfections. Using TB as an example, the analytical and clinical techniques used for diagnosis include sputum smear microscopy and culture, but these methods are inaccurate (microscopy misses 50% of cases) and not rapid (cultures may require days) [62,70] . There is progress being made with the commercial development of NAT platforms that can detect TB infection within a few hours, but how accurate these tests will be when performed on the general population is unknown. Additional challenges facing implementation of these platforms in RLS include the high cost (US$50–60 per test) and feasibility of mobilizing instruments to the field [70,71] . Virus- & vaccine-induced antibodies As the number of large-scale vaccine trials involving complex candidates containing multiple HIV proteins continues to increase, there is the task of accurately differentiating between virus-induced and vaccine-induced antibodies in patients [72] . Vaccine recipients generate antibodies similar to those produced by the humoral immune response following HIV infection, and thus recipients of vaccine candidates may generate false-positive www.futuremedicine.com
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Perspective | Wong & Hewlett results on diagnostic tests that target anti-HIV antibodies [73] . This erroneously labels uninfected individuals as infected and will be a major public-health concern if mass immunizations are administered. Consequences include: the inability to assess the status of a vaccine recipient (and among those truly infected, it cannot be discerned whether exposure came before or after vaccination); rejecting usable blood donations from those vaccinated but uninfected; anxiety and unnecessary care associated with persons who test positive when in fact they are not (which also increases the burden on the medical system); and social stigma or economic discrimination associated with a false HIV status. Diagnostic tests at clinics, blood banks and vaccination sites that distinguish between true virus- and vaccineinduced antibodies will be essential. A test that identifies the conserved epitopes not included in vaccine trials is currently in development to address this need [72,73] . The assay sensitivity is comparable to antibody-detecting rapid tests [74] , which is encouraging. Organ & tissue transplantation Another concern is infection through organ and tissue transplantation. Although rare, there have been reported cases of HIV transmission through organ donations [75,76] . The diagnostic challenges here lie in improving the sensitivity and turnaround times of current tests. While antibody-detecting assays have the shortest turnaround times, they are prone to false-negatives because recently infected donors who are in the diagnostic window period will test negative but can nevertheless infect the recipient. NAT provides the most definitive answer to donor status, but could take up to a day for the results to become available – too slow to be of practical use in common emergency transplant situations [75] . Since a single deceased donor can provide for up to 50 recipients, the screening process includes an epidemiologic history to assess risk, serologic testing, blood and urine cultures, and chest radiography [76] . Nonetheless, these procedures may still fail to detect low-level HIV viremia of those recently infected or undergoing ART. Faster and more cost-effective NAT assays are needed to achieve more stringent testing and prescreening. Since transplants take place in hospitals, an alternative to NAT could be the use of laboratory-based Ag/Ab combo or rapid antigen assays. While not nearly as sensitive as NAT, recent work has shown that nanotechnology-based methods employing gold or europium NPs to detect p24 404
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antigen can improve the level of detection of HIV 100-fold compared with conventional ELISAs and reduce the window period by several days [77,78] . These assays may be used in lieu of NAT assays when immediate results are desired. HIV monitoring & management Assays for HIV monitoring and management provide information about the strains that are present in a given population and patient response to therapy (or lack of therapy). Monitoring
strain diversity & drug resistance Antiretroviral therapy prolongs symptom-free survival by suppressing viral replication rates, but it does not cure HIV. At present, there are over 30 US FDA-approved antiretroviral drugs on the market and over 100 approved or tentatively approved antiretroviral formulations available in association with the US President’s Emergency Plan for AIDS Relief [202] . Owing to initiatives to expand access to ART led by the WHO, the UN, the USA and other partner organizations [79] , the distribution of anti retroviral drugs to patients in RLS has increased from approximately 400,000 people in 2003 to more than 3 million people in 2009 [32,80] . However, drug resistance is a significant obstacle to successful treatment regimens and complicates patient management [58,81–83] . Genetic diversity of HIV results from an errorprone transcription process that introduces numerous substitutions and insertions, as well as recombinations during each cycle of infection. The genetic diversification produced by these mutations confers a selective advantage for HIV to escape the inhibitory effects of drugs [58] . ART regimens usually combine three medications from the different classes of antiretroviral drugs (i.e., nucleoside and non-nucleoside reverse-transcriptase, protease, fusion, entry and integrase inhibitors) to������ minimize escape from drug effects, as mutations that confer resistance to one drug still leaves HIV vulnerable to the others. That is, the probability that drug resistance develops is much lower when multiple inhibitors act simultaneously because multiple escape mechanisms that rely on distinct mutations would be required [80,84] . Treatments that are successful in suppressing viral replication to low levels minimize mutation rates, but even under ideal conditions it is likely that resistance will eventually develop and a growing concern is the emergence of future science group
HIV diagnostics: challenges & opportunities strains nonresponsive to ART. Therapy is usually robust against drug resistance when there are multiple drugs available to cater to individual infections, the patient’s response to treatment is routinely monitored and the drugs are taken according to schedule. The challenge is that RLS are faced with a limited selection of drugs, understaffing and limited health services, which hinder patient monitoring and may result in increased nonadherence to treatment schedules [5,79,85] . When resistance develops against first-line treatments, patients must seek a second-line regimen that is less available and potentially more toxic [1,3,86] , and there are few third-line therapies in RLS [79] . In certain regions the drugs that are available are ineffective against the endemic strains [87] . The choices are even more limited for the pediatric population and up to 69% of mothers can acquire drug resistance when nevirapine, a standard drug given to prevent maternal transmission in RLS, is administered [88–90] . Minimizing the emergence of drug resistance requires an ample supply of newer second- and third-line drugs, and patients who follow treatment schedules. Genotyping assays detect the presence of drug-resistant HIV strains and determine coreceptor tropisms through gene sequence analysis. Whereas genotyping is becoming common practice in resource-rich countries, it is too costly to fully implement in developing countries [85,91] . Standard genotyping methods detect mutants of dominant species or give a composite of HIV sequences present but are generally unable to detect low frequency mutants that comprise less than 20% of the total HIV population [92,93] . Ultra-deep sequencing (UDS) technologies, such as pyrosequencing, polymerase-based sequencing-by-synthesis and ligase-based sequencing, have the ability to generate data on hundreds of millions of base pairs within a few days [94,95] , characterize genotypic diversity, analyze tropisms, and accurately discern rare and drugresistant strains circulating in the population that could potentially cause treatment failure after ART is initiated [96,97] . In studies aimed at detecting and characterizing HIV variants, UDS methods were able to detect a greater proportion of low frequency sequence variants when compared with conventional PCR sequencing methods [97,98] , and there is evidence that UDS may be able to detect mutations present in only 1–2% of the population [93,94] . Other strategies that utilize allele-specific real-time PCR and hybrid yeast systems are able to quantify drug-resistant future science group
| Perspective
mutants down to 0.1–0.4% (which is ~100-fold more sensitive than standard methods); however, these approaches are limited by the number of mutations they can detect and their proneness to false negatives [93] . UDS platforms are expensive, but sequencing technologies have seen dramatic reductions in price and there may be opportunities for their implementation in RLS over the coming years [94,99] . There is also a need for increased research on drug resistance, database development on non-B subtypes (most sequence information is derived from B subtypes in developed countries), and improved standard operating procedures and quality assurance programs to ensure genotyping assays are properly conducted and interpreted [80] . The WHO has established the HIV Drug Resistance Surveillance and Monitoring Network and the HIV Drug Resistance Laboratory Network initiatives to assess, survey and genotype the HIV populations in African countries with ART programs to monitor the emergence of drug-resistant strains [79] . These networks aim to not only provide standardized public-health tools, technical training and assistance, and laboratory quality assurance, but also to develop genotype panels, databases, testing protocols and research agendas [79] . Over 60 nations have adopted the initiative and this represents an opportunity for the evaluation and minimization of drug resistance in RLS [203] . Furthermore, largescale population sequencing of endemic strains enable datasets to be kept up to date and accurate so that evolving bioinformatics tools can play a greater role in diagnosis and treatment selection for patients [100,101] . In addition to genotyping assays, cell-based phenotyping assays are used to determine drug resistance and tropism [80,102] . Since they provide direct evidence of HIV resistance or activity in the presence of drugs, they are more accurate than genotyping assays, but phenotyping assays are of limited use in RLS because they are very costly, labor intensive and difficult to perform outside of specialized research laboratories [102] . The sequence information derived from phenotyping assays conducted in developed nations can be entered into databases to allow genotyping assays to serve as a surrogate platform. Monitoring
changes in disease predictors Monitoring patients who are on ART at regular intervals to assess CD4 + T‑cell counts, viral loads and the presence of comorbidities is www.futuremedicine.com
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Perspective | Wong & Hewlett important for disease management. The degree to which the immune system is compromised and the extent of immunodeficiency are indicated by counting the number of CD4 + T cells and measuring viral loads in the blood. General guidelines have been adopted for the treatment of adult HIV infection; therapy should be initiated immediately if CD4 + T‑cell counts decline at an annual rate over 100 cells/µl, viral loads exceed 100,000 copies/ml or HIVrelated comorbidities are present [23,103,104] . In the USA, treatment would ideally be started prior to CD4 + T‑cell counts dropping below 200–350 cells/µl. The WHO has also recommended CD4 + T‑cell count thresholds according to age for initiation of ART in pediatrics; 1500, 750, 350 and 200 cells/µl for those aged under 11, 12–35, 36–59 and over 60 months of age, respectively [32] . In adults, an effective therapy would produce at least a tenfold decrease in HIV RNA copies/ml within 4 weeks and viral loads should be suppressed to undetectable levels after 6 months [103,104] . Periodic monitoring every 3–4 months is recommended to detect rebounds in viremia or declines in CD4 + T‑cell count, which may be indicative of poor drug tolerability, the emergence of resistance, noncompliance with treatment schedules, the presence of coinfections or possible interactions with other medication [23] . If the viremia remains elevated despite therapy, a new regimen using another combination of medicines should be administered. However, HIV diversity can impair viral load quantification and different assays can generate different results, which limits the accuracy of monitoring [81,83] . In 2006, Medecins Sans Frontieres organized a meeting between academic experts and end users to identify the key characteristics for a viral load assay to be sustainable in RLS. The general consensus from the meeting was that assays should include simple specimen collection methods, use nonrefrigerated reagents, cost under $8 per data point, consist of benchtop or handheld instrumentation priced under US$1000 that run on rechargeable batteries, require minimal training and generate results in less than 2 h [1] . There have been assays adapted for developing countries [105,106] , but they currently fall short of these recommendations. Whereas NAT will likely remain limited to clinical and laboratory settings for the time being [107] , CD4 + T‑cell counting methods using flow cytometry have become simpler, 406
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easier to use, less expensive and more accessible in RLS [108,109] . In concert with clinical monitoring, a CD4 + T‑cell count can be utilized as a predictor to gauge a patient’s response to ART. Future perspective Microfluidic lab-on-a-chip (MLoC) and labelfree platforms have tremendous potential as diagnostic devices for global health [30,110–112] . MLoCs can automate complex assays normally performed in a laboratory onto miniaturized, portable chips with minimal reagent requirements [33] , and be utilized for sample processing, purification, blood fractioning or even basic PCR [30,113,114] . Antibody, antigen, nucleic acid and cell-counting assays that require multiple reagents, capture molecules, fluid handling and detection (e.g., fluorescence, surface plasmon resonance, surface-enhanced Raman scattering, mass spectrometry, electrophoresis and electrical conductance) modalities can be supported by the devices [115–122] . The challenge of ART monitoring was highlighted previously and there are many MLoCs in development that measure CD4 + T‑cell counts out in the field without the need for sophisticated laboratory infrastructures [33,36,115,123–125] . Rapid NAT assays for viral load determination is advancing, but continues to face problems with complexity [107] . Even formats such as inexpensive paperbased systems are in play and actively explored for use in RLS [111,126] . Label-free platforms are typically single-step immunoassays that do not require a scanner to read the assay result and are amenable to rapid, multiplex detection. Nanowire devices consisting of functionalized silicon wires that are 2–20 nm thick can detect DNA, antibodies, antigens and even whole viruses with femtomolar sensitivities [127] . Each nanowire acts as a field-effect transistor that produces a measurable change in conductance when a binding event takes place on the wire surface, and an array of nanowires can be constructed on a single chip for multiplex detection [128] . In a nanocantilever system, a series of 100–750 µm long × 20–100 µm wide × 0.6–1.0 µm thick functionalized cantilevers act as sensors when changes in mechanical bending and vibrational frequency are identified in the event of binding [129] , and even single-base mismatches in DNA are discernable [130,131] . In 2009, an effort was initiated in London, UK, to develop a nanocantilever device for the bedside detection and monitoring of HIV/AIDS [204] . future science group
HIV diagnostics: challenges & opportunities Another promising platform is the capacitance biosensor, which measures the displacement of counter ions surrounding the capture ligand after a binding event takes place on a functionalized surface [132] . These sensors are able to detect various biotargets of interest [133–136] , and sensitivities in the subattomolar range were demonstrated [137] . Also in commercial development are sensors that recognize antibody–antigen binding and DNA hybridization using surface plasmon resonance [33] . While instrumentation is required to capture assay signals, the label-free platforms described above generate a result within minutes, can be engineered into portable formats, enable multiplex detection and are poised to mature within the next few years. However, MLoCs and labelfree platform are not yet widely used outside of research laboratories and their performance will need to be thoroughly evaluated and validated out in the field using clinical samples. Another concern is whether they will be cost effective, so as to justify their use over current rapid tests. Nanotechnology is rapidly finding a place in many biomedical applications and may become an important component in many diagnostic platforms by replacing many of the existing detection labels [138] . Owing to their ability to scatter light, NPs produce much stronger signals as a label than fluorescent probes, and are thus able to achieve much lower levels of detection [139] . NPs enable the design of ultra-sensitive immunoassays and allow diagnostic devices to generate results that are visible with the naked eye, as in the case with rapid tests [77,78] . With the analytical sensitivity demonstrated by gold, silver and fluorescent NPs, consideration should always be given to NPs when developers design immunoassays. Microarrays have numerous applications in protein detection and are able to analyze thousands of samples in parallel in a short period of time with minimal reagent requirements. In the laboratory setting, microarrays are adaptable to antibody, antigen and DNA assays (among others), able to generate rapid results, conducive to multiple detection schemes, quickly produced and virtually unmatched in multiplexing capability [140–146] . Microarrays have also been deployed for field testing of pathogens, but several obstacles remain [147] . In order to achieve acceptable sensitivity, current DNA and oligonucleotide arrays require sample amplification by PCR, which is undesirable. The accuracy of antibody and antigen arrays depends on capture future science group
| Perspective
quality, and cross-reactivity is a concern given the diversity of HIV. Microarrays that contain hundreds to thousands of features will require a scanner and software analysis to interpret results. The development of a genomic microarray that can target multiple strains, subtypes, and recombinant and drug-resistant forms of HIV would be a significant advance even if only used in research settings. When detection is accomplished through a capture assay or PCR primer, there is the challenge of procuring suitable agents to do the job. The diversity of HIV makes it very difficult to have a standard set of targets apart from IgG/IgM, p24 antigen and certain conserved regions of the genome, although they too are variable. Furthermore, detection based on conserved epitopes provides no information about subtype or recombinant form. Whether it be an open and flexible oligonucleotide or aptamer design strategy or capture by other means, a well-defined scheme to actively design complimentary molecules as detection agents will be necessary to keep d iagnostic tests up to date with circulating strains. Another critical component will be automated sample acquisition, preparation and processing (SAPP) steps at the front end of a diagnostic test to maximize the accuracy and consistency while minimizing human error. Synergy between SAPP and diagnostic assays is vital to achieving optimal results. Magnetic particles are now commonly utilized to purify targets of interest [148,149] , and aptamer-based beads or filters may also play a major role in sample preparation in the near future [150,151] . Evidently, the impact of HIV and its associated public-health complications are a global burden and there is no immediate unified solution to the problem. In the absence of a cure or universal prophylactic, increased efforts in diagnostics development, expanding accessibility to ARTs and education on prevention are critical measures to slow the rate of transmission. Compared with just a decade ago, there are signs that the epidemic in Africa is stabilizing, yet the continued spread of HIV in other parts of the world is evidence that current strategies are still insufficient [201] . International and local efforts to draft tailored programs for individual countries, catering to their unique cultures, behaviors and infrastructures, will be an essential component to tackling the problem. Political (national, community and institutional) and social (families, couples and individuals) hierarchies should harmonize www.futuremedicine.com
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Perspective | Wong & Hewlett to dispel the stigma associated with HIV status, and sufficient human resources should be put in place to adequately run healthcare programs. It is up to researchers and developers to produce the diagnostic platforms that will address the cost, accuracy, rapidity, portability, multiplexing and simplicity needs for RLS. An important general point is that gradual improvements of current technologies will have a beneficial impact and emphasis should not necessarily be placed only on designs that address worstcase scenarios, which ultimately could add cost and complexity. Furthermore, while deployable tools, such as portable devices and handheld analyzers, are sought for RLS, a lot of research is aimed at proof-of-concept platforms with limited consideration of how to bring them to market for actual use. Rather than focusing solely on the technical aspects and performance of the diagnostic, commercial developers may wish to also communicate with public-health officials to better understand the environments, protocols and legal frameworks within which the tool will operate to maximize their practical usefulness. Collectively, the technological and procedural
advances of the next few years may produce an era in the 21st century when HIV diagnostics can be made affordable and accessible to all. Disclosure The findings and conclusions in this article have not been formally disseminated by the US FDA and should not be construed to represent any Agency determination or policy.
Acknowledgements We would like to thank Drs Shixing Tang, Pradip Akolkar, Andrew Dayton, Krishnakumar Devadas, Hira Nakhasi and Ginette Michaud for their comments and review of the manuscript.
Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Executive summary Global burden of HIV & its management HIV continues to evolve, develop drug resistance and spread worldwide. There is a need for increased accessibility to accurate diagnostic tests and antiretroviral drugs for those living in high HIV-prevalent settings in order to minimize transmission rates. Challenges facing HIV diagnostics (& patient management) The rapid tests that are becoming widely available in resource-limited settings (RLS) require quality assurance programs for evaluation and monitoring. The most commonly used tests detect anti-HIV antibodies and are suboptimal for infant and adult acute infection testing. Accurately distinguishing between recent and long-standing infections is important for monitoring HIV transmission patterns, but current incidence assays are prone to error. The genetic diversity of HIV can produce false or discordant results on ELISA and nucleic acid testing platforms. Testing for common HIV coinfections is very limited in RLS. Diagnostic tests that can distinguish between vaccine- and HIV-induced anti-HIV antibodies will be needed in communities where large-scale vaccine trials take place. Screening tests need to be more sensitive and have shorter turnaround times to ensure organ and tissue transplants are free of HIV. While antiretroviral therapy has greatly reduced the mortality rates of those infected with HIV, developed drug resistance is a concern. Sequencing and genotyping approaches used to determine drug-resistant and low-frequency strains are limited in RLS. Patient response to therapy is usually monitored through viral load levels and CD4+ T-cell counts, measurements that are difficult to ascertain in individuals at the point of care. Opportunities & future perspectives Microfluidic lab-on-a-chip, label-free and antibody/antigen combination platforms hold promise as the next generation of diagnostic tools. As the amount of sequence information and size of HIV repositories increase, stakeholders will have better tools from which to develop improved and more accurate diagnostic tests. Ultra-deep sequencing technologies have the ability to detect drug-resistant and low-frequency HIV strains. The WHO is leading an initiative to genotype and monitor drug resistance in RLS. Nanoparticles used in place of fluorescence-based modalities can improve detection sensitivities. Microarrays can address the need for multiplexed diagnostic tests, but high-quality capture agents will be required. Developers and health officials should communicate to ensure diagnostics have optimal usefulness in real-world settings and researchers should focus on how to bring their novel discoveries to market for actual use.
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