Point of Care Technologies for HIV

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Review ArticlePoint of Care Technologies for HIV

Mohan Kumar Haleyur Giri Setty and Indira K. Hewlett

Laboratory of Molecular Virology, Division of Emerging ransfusion ransmitted Diseases,Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD , USA

Correspondence should be addressed to Mohan Kumar Haleyur Giri Setty; [email protected]

Received April ; Accepted November ; Published January

Academic Editor: Bryce D. Smith

Copyright © M. K. Haleyur Giri Setty and I. K. Hewlett. Tis is an open access article distributed under the CreativeCommons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided theoriginal work is properly cited.

Effective prevention o HIV/AIDS requires early diagnosis, initiation o therapy, and regular plasma viral load monitoring o theinected individual. In addition, incidence estimation using accurate and sensitive assays is needed to acilitate HIV preventionefforts in the public health setting. Tereore, more affordable and accessible point-o-care (POC) technologies capable o providingearly diagnosis, HIV viral load measurements, and CD counts in settings where HIV is most prevalent are needed to enableappropriate intervention strategies and ultimately stop transmission o the virus within these populations to achieve the uture goalo an AIDS-ree generation. Tis review discusses the available and emerging POC technologies or uture application to theseunmet public health needs.

1. Introduction

Acquired immunodeciency syndrome (AIDS) is a diseaseo the human immune system caused by the HIV [] whichdestroys CD+ lymphocytes o the immune system thatprevent inections. AIDS is one o the most serious globalhealth problems o unprecedented dimensions and is oneo the greatest modern pandemics. At the end o , anestimated million people were living with HIV globally,including . million children under years o age. Terewere . million new HIV inections in , including

among children less than years [].Te annual number o people newly inected with the

HIV has declined % rom the global epidemic peak in, mainly due to tremendous progress in diagnosticsand treatment. Several diagnostic technologies have emergedwith high specicity, sensitivity, and accuracy to detect HIVinection. HIV testing plays an important role in HIV preven-tion in that knowledge o HIV status has both individual andpublic health benets. Early and accurate detection o HIVinection is important to public health because this stage ischaracterized by high inectiousness and transmissibility o the virus. Te individual benets o HIV testing are primarily associated with individuals accessing care and treatment.

Individuals entering care and treatment have a substantialreduction in adverse health outcomes and increased lieexpectancy.

Te past decades have witnessed enormous technologicalimprovements towards the development o simple, cost-effective, and accurate rapid diagnostic tests or detectionand identication o inectious pathogens. Tere is growingdemand within the global health community to nd waysto simpliy and improve the efficiency o diagnostics orHIV/AIDS without diminishing the quality o patient care.At the same time, there is a need to signicantly increase the

level o access to robust, high-quality diagnostics in resource-limited settings in order to acilitate HIV prevention throughearly detection and treatment. Low-cost technologies todiagnose and monitor HIV inection in developing countriesare a major subject o current research and health carein the developing world. With the great need to increaseaccess to affordable HIV monitoring services in rural areaso developing countries, much work has been ocused onthe development o point-o-care (POC) technologies thatare affordable, robust, easy to use, portable, and o sufficientquantitative accuracy to enable clinical decision making. Fordiagnosis o HIV inection, some low-cost tests, such aslateral ow tests and enzyme-linked immunosorbent assays,

Hindawi Publishing CorporationAIDS Research and TreatmentVolume 2014, Article ID 497046, 20 pageshttp://dx.doi.org/10.1155/2014/497046


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AIDS Research and reatment

are already in place and well established. However, portablequantitative tests or rapid, POC, HIV monitoring have only recently been introduced to the market.

Tere is great need or initial diagnosis, staging, andongoing monitoring o HIV. Among those that presentthe most persistent challenges to improved access and

efficiency are CD, viral load, and early inant diagnosis(EID). Te great majority o testing options available today are laboratory-based platorms perormed on sophisticatedinstrumentation requiring dedicated laboratory space andtrained technicians. In many cases, laboratory-based test-ing is expensive; in almost all cases, it requires specimentransport networks to enable access or patients in periurbanand rural settings. Te success o high-quality antiretroviraltherapy (AR) depends on simple, affordable, reliable, andquality-assured POC diagnostics or use in resource limitedsettings (RLS). POC diagnostics can make AR more scalableand will allow AR service delivery to be signicantly decentralized to the community level. At the same time,simpliying diagnostic technologies that could potentially reduce the cost o diagnosing and monitoring patients livingwith HIV/AIDS without diminishing the quality o care isimportant or treatment success. Te World Health Orga-nization (WHO) has set criteria or at or near POC use—known as “ASSURED” criteria, meaning that they are (or willbe) affordable, sensitive, specic, user-riendly, robust/rapid,equipment-ree, and deliverable to those who need the test[].

POC diagnostics are in vitro diagnostics (IVD) that do notinvolve the use o laboratory staff and acilities to provide theresult. Te analytical “targets” include proteins, nucleic acids,metabolites, drugs, dissolvedions andgases, human cells, andmicrobes. est samples can be blood, saliva, urine, or otherbodily uids or (semi-) solids. Whether used “near patient”in a hospital, clinic, or doctor’s office or administered at hometo maintain health, manage disease, or monitor therapy or inthe eld to test the saety o water, ood, or compliance withlaws and regulations, these tests accept a sample with little orno preparation and provide a result, the “answer,” in secondsto hours [].

Te tests should require only elementary instruction touse and some detect multiple analytes or markers. Interpre-tation should be as simple as viewing a stripe or spot o coloron a strip o paper or polymer; increasingly, however, readersranging rom hand-held devices to bench-top instrumentsread the analytical test, provide a comprehensible result,

and i necessary, control and operate the sample-containingplatorm that executes the analytical process. Tese devicesshould be capable o using nanoliters to milliliters o complexbiological media with emtomolar to millimolar concentra-tions o analytes []. Te devices should be inexpensivedisposable units or cartridges that include microuidic ea-tures to provide or control sample preparation, ow rate,mixing with reagents, reaction time associated with bindingevents, ltration o nonanalytical components o the sample,separation o interering agents and o multiple analytes, andan effective measurement capability [].

“Why POC Diagnostics or HIV?” POC measurementsprovide results rapidly, where needed, and ofen with major

time savings, samples do not travel to a laboratory to awaitthe attention o a skilled technician; results do not wait tobe transmitted and collected. Rather, the doctor, nurse, care-giver, patient, or consumer initiates the test and receives theresults on the spot. Inevitably, this saves time, but speed mustnot be traded or accuracy or reliability.

2. HIV Diagnostics

Diagnostics or HIV/AIDS can generally be divided intothree test categories: (i) tests to acilitate initial diagnosis,(ii) tests to stage the patient, and (iii) tests to monitor thepatient, both beore and afer initiation o AR. Tere aregenerally accepted algorithms and tests used at each stage o the inection [].

.. Low-Cost Diagnostic ools for Monitoring HIV. Low-costtechnologies to diagnose andmonitor HIVinection in devel-

oping countries are a major subject o current research inhealth care settings in the developing world. With increasingneed to provide access to affordable HIV monitoring servicesin rural areas o developing countries, much work has beenocused on the development o point-o-care technologiesthat are affordable, robust, easy to use, and portable and o sufficient quantitative accuracy to enable clinical decisionmaking. For diagnosis o HIV inection, some low-cost tests,such as lateral ow tests and enzyme-linked immunosorbentassays, are already in place and well established. However,portable, quantitative tests or rapid HIV monitoring at thepoint o care have only recently beenintroducedto the market[]. Te Ora Quick Rapid HIV-/ Antibody est is a lateral

ow rapid test or oral uid specimens that perorm as well asblood-based tests,even at lowconcentrations o HIVantigensin oral uid []. Te Aware HIV-/ U, with .% sensitivity and % specicity, is a rapid alternative to urine tests thatrely on ELISAs and Western blots.

Rapid HIV tests are expanding to include combinationtests that detect both anti-HIV antibody and p antigen, aswell as tests or early inant diagnosis and or HIV- and HIV- subtype differentiation []. Detection o both antibody andantigen during the acute phase o inection is particularly benecial to prevention efforts because the rate o HIVtransmission is several olds higher during this phase and canaccount or up to % o new HIV inections. Te DetermineHIV-/ Ag/Ab Combo is an immunochromatographic rapidtest that detects antigen and antibody separately. In twoindependent studies, the Determine HIV-/ Ag/Ab Comboshowed sensitivities o % and % or antigen detection[, ].

.. HIV Viral Load Monitoring. HIV viral load monitoringis crucial or therapy and staging o the disease. Reverse tran-scriptase (R), an enzyme present during viral replication,correlates linearly with HIV load and CD+ -cell count [].Measuring this R protein may be an alternative or resourcelimited settings, as its yielding results in a ew hours and costs% less than nucleic acid tests [].


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Viral load estimates based on reverse transcriptase testsare not directly comparable to those obtained through PCR because they have been ound to underquantiy the HIVload [, ]. However, in places where PCR is unavailable,results o reverse transcriptase tests can be a cheaper viralload monitoring method. Further advantages o reverse tran-

scriptase tests include their ability to detect multiple subtypeso HIV and their possible use in monitoring HIV load inpediatric patients []. Levels o the HIV coreprotein p alsocorrelate with HIV load and CD+ -cell count. Assays orp have been shown to have lower sensitivity and there hasbeen difficulty in correlating plasma p and viral RNA loadin persons receiving AR []. However, improvements insensitivity o p antigen tests may allow urther reevaluationo these assays or HIV viral load monitoring.

... Current Viral Load echnologies. HIV viral load tech-nologies can be categorized broadly as nucleic acid-basedtests (NA) and non-NA-based technologies. NA tech-

nologies detect and quantiy viral RNA, whereas non-NAtechnologies detect and quantiy HIV viral enzymes andproteins that can be correlated with the amount o viralRNA. Most o the commercial assays use target amplica-tion methods []. In general, the advantages o NA-basedapproaches are their wide availability in quality-assured kits,and clinicians have amiliarity with interpreting the results.Te assays vary in terms o sample preparation andamplica-tion/detection methodologies. Te major NA-based assaysand platorms are discussed below [].

Tere are our commercially available R-PCR based viral load assays at present: (i) COBAS AMPLICOR HIV- MONIOR v. (Roche Molecular Systems), (ii) COBAS

AmpliPrep/COBAS aqMan v. (Roche Molecular Sys-tems), (iii) Real-ime HIV- (Abbott), and (iv) VERSANHIV- RNA . assay (kPCR) (Siemens). Tere are also anumber o other in-house procedures and test systems thathave good sensitivity and reproducibility and are used in

various countries. Te non-NA-based technologies quantiy proteins and enzymes specic to HIV. Tese include assaysthat measure the level o reverse transcriptase activity andassays that measure the concentration o circulating pprotein [].

... Reverse ranscriptase echnologies. Reverse transcrip-tase (R) assays detect that viral enzyme, the R activity can

be quantied, and levels can be correlated with the amounto HIV. Tereore, an assay or R can reect the HIV viralload in the patient’s blood. R assays originally requiredradioisotopes, a scintillation counter, and an ultracentriugeor perormance, but they have been simplied and made lesshazardous. Currently, there is one R platorm available orin vitro use—the ExaVir Load, manuactured by Cavidi AB[, ].

.. ExaVir Load (Cavidi AB). Cavidi manuactures theExaVir (Version ), which is a quantitative HIV-R test that isdesigned to measure viral-bound HIV R activity in plasmain order to estimate the HIV viral load. Te ExaVir Load

assay is more manual than most o the other viral load assaysdescribed herein, but it is generally less expensive than othercurrent molecular detection methods.

An advantage o the assay is that because the ExaVirLoad determines viral load based on quantication o Ractivity and does not target a specic nucleic acid sequence;

it can measure any HIV type or subtype with high accuracy,including O and N groups. Te measuring range o the assay is the equivalent o about to , copies/mL (or to , emtograms g/mL). Tere is perormance dataavailable on the ExaVir Load showing good correlation withthe COBAS AMPLICOR Monitor assay [, ].

... p Antigen echnologies. HIV- inection is charac-terized by an early spike in HIV- antigens in the blood.During this period o acute inection or antigenemia, theantigens in the blood are detectable, but in most individuals,the antigen levels subsequently become undetectable or aperiod o time. It is only later in HIV disease, with increasingailure o the patient’s immune system and an increasinglevel o the virus, that the antigens may again becomedetectable in the blood. One o the viral components inblood during the period o antigenemia is the core protein,p, the major internal structural protein o HIV-. Te pappears within weeks afer inection as a result o theinitial increase in viral replication and is associated withthe period o antigenemia during which the individual ishighly inectious. esting or p antigen can be o value inseveral circumstances: (i) detecting early HIV inection, (ii)diagnosinginectionin inants (discussed later in thisreport),and(iii) monitoring AR. In the past, beorethe availability o NA-based technologies, the p antigen assay was used or

monitoring the development o AIDS and monitoring diseaseprogression []. In particular, the NEN HIV- p ELISAassay rom Perkin Elmer (an ultrasensitive, heat denaturedp antigen quantication assay) has been used or thispurpose. However, the p antigen tests available were not

very sensitive and there are concerns about correlation o pwith HIV RNA []. Moreover, with a linear range between, and , RNA copy equivalents/mL, the assay is o limited utility in detecting early treatment ailure and it is notuseul in patients with low viral replication []. Tereore,the use o p antigen testing will not be discussed urther inthis report in the context o monitoring patients on AR butwill be revisited in the discussion o EID.

Te latest laboratory immunoassays to come to themarket or central laboratory use are antigen/antibody immunoassays, which can detect p antigen in additionto detecting anti-HIV, IgM and IgG. Combination anti-gen/antibody assays have been approved and used in many countries since the late s [–]. Tese assays detect pantigen at the level o – pg/mL [], which is equivalent toapproximately ,–, copies/mL o HIV RNA [].Recently, two such assays (Abbott ARCHIEC HIV Ag/AbCombo and Bio-Rad GS HIV Combo Ag/Ab EIA) have beenapproved or use in the USA. Several studies have beenconducted with these assays and the data indicate that they have similar perormance characteristics as those marketed


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elsewhere [–] and detect p approximately – daysafer the appearance o nucleic acid []. Although these assaysare central lab-based assays, they can be adopted to POCplatorms oruse in resource-limited settings as theyare moresensitive and diagnose HIV inection earlier with a higherdegree o accuracy.

... Viral Load echnologies for Future Use. Te aboveNA-based viral load systems described require testing tobe perormed in a laboratory setting at a central or nationalreerence laboratory, by well-trained technicians. Althoughthere are currently no POC viral load assays on the market,there are a number o platorms/assays in development.Described below are a ew viral load assays that are currently being used and in the pipeline.

.. NA System (Alere Inc.). Te Alere NA system is ageneric platorm or the implementation o different NAassays with an integrated platorm or quantitative measure-

ment o HIV- and HIV- viral load rom approximately L o whole blood. Te system detects HIV- Groups M,N, and O and HIV-. Te device on which the assay is run (aprototype o which is pictured below) has a small ootprint,is portable, battery-operated, and ruggedized to withstandharsh environments [, ].

.. Liat Analyser (IQuum, Inc.). Te Liat Analyser, manu-actured by IQuum, Inc., is an automated sample-to-resultNA platorm that perorms sample nucleic acid extraction,purication, reverse transcription, polymerase chainreaction(PCR) amplication, and real-time detection to detect and/orquantiy pathogens. Liat assays or HIV viral load testing

and diagnostics have also been developed and independently evaluated by several laboratories. o aid the operator andprovide reliable results, the Liat Analyser incorporates a vari-ety o advanced eatures: barcode data entry avoids errors insample or assay coding and on-screen prompts provide easy-to-ollow directions to guide the operator through sampleloading and tube insertion [].

.. EOSCAPE-HIV HIV Rapid RNA Assay System (Wave Biosciences). Based on its EOSCAPE technology, Wave Biosciences is developing a rapid HIV NA-based POC

viral load assay designed or use in resource-limited settings.Te company describes the assay technology as incorporating

non-PCR nucleic acid detection and quantitation, ngerstick whole blood processing within a single-use, enclosed car-tridge. Using a ngerstick lancet, L o whole blood isapplied directly into the cartridge and no external samplepreparation is required. Te sample is automatically pro-cessed in minutes; the operator then inserts the processingunit into the reader or a quick -minute scan. Equipped witha simple touchscreen interace, the reader is capable o trans-mitting test results through wired and wireless connectivity.Te EOSCAPE-HIV system will be capable o providingeither a qualitative or a quantitative HIV- RNA test result(detection threshold o , copies/mL) in less than an hour[].

.. SAMBA (Diagnostics for the Real World). Te SimpleAmplication Based Assay (SAMBA) is being developed by the Diagnostics Development Unit (DDU) at the University o Cambridge. wo NA-based HIV assays are being devel-oped: (i) a semiquantitative test or monitoring o AR and(ii) a qualitative test or the use in EID. Te rst SAMBA

HIV assay to be launched will be the semiquantitative viralload assay. Te SAMBA machine will integrate extraction,amplication, and detection into a bench-top analyzer withamplication and detection taking place in a closed cartridge[].

Te SAMBA HIV test uses L o plasma or Lo whole blood. Amplication is based on both target andsignal amplication. Te visual detection o the RNA orDNA target can be read off o a test strip visually within minutes. Te test strip is based on a nitrocellulose membranein a dipstick ormat. Te SAMBA assay was able to detectall HIV- subtypes at cp/mL. Currently, however, theSAMBA semiquantitative assay is calibrated to distinguishbetween patients with viral loads above or below cp/mL.Te total amplication time o the SAMBA is one hourwith throughput suitable or use at a small laboratory—or example, at district hospital level in sub-Saharan Arica.Diagnostics or the Real World, Ltd, the spinout company o DDU located in Caliornia, will be the manuacturer o theSAMBA system [].

3. Additional Viral Load Technologiesin the Pipeline

Te Cepheid GeneXpert System, a ully automated andintegrated system or PCR-based nucleic acid testing, is

in use or detection o multiple pathogens and is beingdeveloped or HIV. Te Northwestern Global Health Foun-dation (NWGHF) in collaboration with Quidel Corporationis developing a POC rapid R-PCR testing platorm that willbe both easy to use and o low cost. Lumora has introducedthe Bioluminescent Assay in Real-ime (BAR), a platormor perorming molecular diagnostics that allows real-timeclosed-tube quantitative detection o amplication by usinga hardware system that can generate and store objectivetest results. Advanced Liquid Logic, Inc. (ALL) providesdigital microuidics technology solutions or liquid handlingapplications. Te company has developed a compact bench-top immunoassay analyzer (pictured right) that is currently

being evaluated. Te company indicates that it is evaluating various assay ormats as well as the portable analyzer. Cavidiis developing a new automated platorm or near-patient viralload monitoring. Te system will combine the strengths o R technology with the advantages o an automated walk-away platorm. Tis should provide ast and robust viral loadmonitoring or all HIV types and subtypes [].

.. Early Infant Diagnosis. Diagnosing HIV inection early in inants can help in suitable therapeutic intervention whichgreatly reduces inant mortality due to HIV inection. Cur-rent p immunoassays have improved sensitivity comparedwith previous p tests because o the inclusion o a heat


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denaturation step or separating p-antibody complexesthat commonly interere with assay sensitivity. Many studiessupport the use o the Perkin Ultra p immunoassay anddried blood spot analysis or early inant diagnosis (EID).However, p immunoassays are not widely used owing toa lack o validation data. Also, because p immunoassays

require specialized equipment and skilled staff, p rapidtests are preerred or EID in resource-limited locations[]. Development o p rapid tests has ocused mainly on improving sensitivity []. In , Parpia et al. testeda microuidic p test on L plasma samples rom South Arican inants []. Teir design included a heat-shock component to break the antigen-antibody complexes.Tey achieved % sensitivity, % specicity, and a detec-tion limit o RNA copies/mL. Te current prototypecan be perormed within minutes.

.. Ultrasensitive p Antigen Assay (NWGHF). NWGHF isdeveloping an ultrasensitive p antigen rapid lateral ow

assay or POC use. Te technology, to be called Lynx, involvesnot only a lateral ow strip that detects HIV p antigen butpreanalytical devices or separating plasma rom heel-stick blood and disruptingimmune complexes thatwould intererewith immunoassays. NWGHF has demonstrated proo o principle o the test. Te assay procedure involves collectingL o heel-stick blood rom the inant using a capillary tube; plasma is separated and the sample is subjected to “heatshock” in small, battery-powered processor device; the rapidtest strip is inserted into the device; and afer approximately minutes the result is read. Te total assay duration isapproximately minutes. In early testing, the assay hasshown about % sensitivity and % specicity.

.. PanNA Diagnostic Platform (Micronics, Inc.). Micron-ics, Inc., a subsidiary o Sony Corporation, has developed thePanNA system, which is a small, portable microuidic plat-ormorPOC use in in vitro molecular diagnosis o inectiousdiseases in resource-limited settings. It uses a uorescent-based reader capable o processing individual, disposable,assay-specic cartridges, each o which is designed to per-orm a single- and/or multiplexed nucleic acid assay. Tereader includes all necessary reagents on board. Te systemis lightweight, portable, battery- or centrally powered., WiFi-enabled, capable o storing up to test results beoreprompting the user to download or delete results, and can

provide results within to minutes.

Portable ests. Portable tests or HIV monitoring compriseCD+ -cell count assays and HIV quantication assays.Currently, only a handul o truly portable CD+ -cellcounters are available commercially: PointCare NOW, theCyFlow miniPOC (Partec), Pima est (Alere), and DaktariCD+ (Daktari Diagnostics). All are ully automated, can bepowered by batteries or electricity, use ≤ L o blood, andprovide same-day results. Additional POC CD+ -cell testsin development include a multiplex inectious disease test(MBio Diagnostics) and a semiquantitative, electricity-reeCD+ -cell blood test (Zyomyx) []. Both the PointCare

NOW and CyFlow miniPOC measure absolute CD+ -cellcounts and the percentage o cells expressing CD+ anddo not require cold chain or reagent storage. Te CyFlow miniPOC has a substantially high throughput, with thecapability o processing tests per day. No independentdiagnostic data or either test were available at the time o

writing. Te Pima estand Daktari CD+ are cartridge-basedtests in which a blood sample is inserted into a disposablecartridgecontaining the necessary reagents per test. Te Pimaest, introduced in late , has yielded results comparableto those o other AR monitoring devices [–]. While themajority o Pima est measurements underestimate CD+-cell counts, one study demonstrated .% sensitivity orconcentrations below a cut-off o cells/L []. A recentstudy also ound that Pima est capillary blood samplesyielded less precise results than venous blood samples. Tishighlights the need to address sources o error that candecrease a POC test’s accuracy. No portable PCRtestsor HIVquantication are currently on the market but several are inthe pipeline.

Several other POC tests that may be portable are alsounder development. According to PAH, a low-cost versiono a ully automated rapid PCR device is in developmentor resource-limited settings []. Tis test, known as theLiat HIV Quant Assay (IQuum Inc., Marlborough, USA),uses whole blood in a single container or PCR amplicationand analysis in minutes []. Te SAMBA HIV- testis a dipstick-based nucleic acid assay or HIV detectionand amplication. Afer collection o viral RNA, isothermalamplication o RNA is automated by a machine. Te currentprototype tests one sample in


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HIV prevention []. In addition, the noninvasiveness o specimen collection eliminates the anxiety associated withblood collection and leads to higher rates o voluntary HIVtesting [].

.. CD Counts. Te measurement o the absolute CD -

cell count is critical in the initial evaluation and staging o HIV-inected persons. HIV inects primarily immune CD+ lymphocytes (CD+ cells) []. Antiretroviral therapy,AR, especially when CD+ cells are not yet depleted,can reduce viremia and slow its progression to AIDS [].Furthermore, i CD counts are established, antiretroviralprophylaxis can reduce the risk o transmission in pregnantwomen to prevent mother-to-child transmission o HIV dur-ing pregnancy, childbirth,and breasteeding []. Tereore itis critical to perorm CD+ cell countsupon diagnosis o HIVand beore initiating AR. Te WHO currently recommendsAR initiation i absolute CD counts are below /cellsmm []. Te perormance o the diagnostic platorm with

respect to accuracy and precision is the most important orusing such technologies. Tis is particularly challenging orCD testing platorms as “no gold standard technology orinternationally recognized reerence preparation exists orCD” []. Misclassication may delay the initiation o ARor prophylactic treatment in some patients or treat largenumbers o patients who have CD counts above the ARinitiation threshold.

.. Existing CD echnologies/Platforms. Tere are currently a handul o platorms that account or CD testing inresource-limited settings. Tese are lab-based single platormsystems rom BD Biosciences, a division o Becton Dickinson(BD), Beckman Coulter (Coulter), Millipore (ormerly Guavaand now a division o Merck), Partec, and Apogee. In thedeveloping world, BD and Coulter are the most commonly used platorms or CD testing [].

.. Point of Care CD esting Platforms. Te above-mentioned high-, medium-, and low-throughput platormsare systems primarily designed or use in laboratory settings.A number o them, including the FACSCalibur, Epics andFACSCount, are used in developed as well as resource-limited settings. Te CD assays developed include selec-tive cell staining, ollowed by capture or count by digital

photography, measuring CD molecules instead o cells, ormeasuring proxy molecules o CD. Some o the POC tech-nologies are already in use. Tey include PointCare NOW,the Pima CD Analyser, and the CyFlow CD miniPOC.Te remaining technologies including Daktari, Burnet,Zyomyx, MBio, and others are not yet commercially available[].

.. Other Possible CD POC ests. In addition to thePOC CD tests/devices discussed above, there are a ew other research and development groups working on plat-orms/devices that could potentially be used or CD count-ing.

.. Automated POC CD est Devices. Tere are severalcommercial POC CD counting devices that are now avail-able and some are under development. Most o them usenger-stick blood samples, which are rapid, are robust, haveexible power options, and utilize stable, dried reagentsenclosed in a single test cassette or device. Te devices are

designed or minimal operator use with varying through-put result outputs. Te PointCare NOW and the CyFlow miniPOC are modiedow cytometers. Te PointCare NOWis the only POC CD device commercially available. Tesystem is signicantly heavier than the other technologiesmaking it less portable andvenipuncture is required orlargerspecimen volumes, which may limit its use in low resourcesettings where phlebotomists are scarce. However, additionalequipment such as pipettors is not needed or operation andthereore supply o other critical reagents is not a actor.

A CD POC test marketed by Alere (Waltham, MA), thePima CD test, is the rst commercially available automatedCD counting method that does not use traditional ow cytometric approaches. Te Pima utilizes dual-uorescenceimage analysis to count CD+ and CD+ using labeledanti-hCD and anti-hCD. Te Pima cartridge contains allreagents and specimen during processing. Te Pima readersupplies the pumping unctions and images the results todetermine colocalization o CD+ and CD+ cells. Te resultexpressed as the absolute number o CD+ lymphocytes perL and the CD+/CD+ ratio is displayed by the instrumentalong with quality control results. Cartridges using beadsthat represent “Normal” and “Low” counts are intended ordaily quality control testing o the PIMA instrument. Recentevaluations in Zimbabwe and Mozambique have shown goodperormance in comparison to ow cytometry and userassessment o the test protocols that suggest that the Pima hasthe potential or use at the POC [, ].

Daktari Diagnostics, Inc. (Boston, MA) is develop-ing a CD test that uses a novel microuidic affinity-chromatography/shear-gradient technique to differentially capture CD+ cells rom whole blood and a unique nonop-tical detection to count them. Te test consists o a readerand a disposable cassette which contains all o the reagents,stabilized in blister packs. Here, owing CD+ cells adhereto an antibody-coated chamber while larger-sized monocytesare subject to large shear orcesand cannotbind. Te capturedCD cells are then lysed and the release o cellular ions ismeasured by impedance spectroscopy []. Te decrease inimpedance in the chamber due to CD+ cell binding has

been shown to correlate with cell count over three orders o magnitude, including CD+/mm, the threshold or AR[].

mBio Diagnostics, Inc. (Boulder, CO) is developing aCD+ cell-counting system, SnapCount. It is a static two-color uorescence imaging cytometry system composed o single-use disposable cartridges and a simple reader withon-cartridge immune staining o whole blood samples. Teinstrument addresses the high cost o conventional opticalsystems by using LightDeck technology, a uorescence assay illumination approach that is a variation on planar waveguidetechnology which uses low-cost lasers, optics, and imagingsensors that are now ubiquitous in cell phones and consumer


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electronics. As the instrument is only utilized in the read-ing step, multiple cartridges can be processed in parallel,providing a throughput o eight to ten samples per hour.Tis relatively high sample throughput might allow remotehealth care settings to meet their greater demand with ewerinstruments [].

.. Nonautomated CD est Devices. In , the CDinitiative held by Imperial College, London, (unded by theBill & Melinda Gates Foundation) partnered with industrialand academic teams to develop power-ree POC CD tests[] and three devices are now being urther developedrom this project. Te Burnet Institute CD test device is asemiquantitative immune chromatographic strip(ICS). SomeICS assays have been shown to be effective tools or POCdiagnostics in RLS; theyare small, o lowcost, have acceptableperormance and reagent stability, and have a test ormatthat is amiliar to the intended user group. Te Burnet CDtest has been incorporated into a lateral ow strip (similar

to an HIV rapid diagnostic test) with traditional rapid testormat, including monocyte removal pad and immunogold.In this device the transmembrane domain o cellular CDis recognized by biotin-labeled anti-hCD which is detectedby a colloidal gold labeled anti-biotin. A capture stripe o anti-CD is adjacent to a reerence stripe o a biotinylatedsurrogate protein. Te CD count is determined by the userto be either greater or lower than the reerence stripe.

Beckman Coulter has been developing a similar semi-quantitative ICS ormat to enumerate CD at the POC.Zyomyx, Inc. (Hayward, CA) has developed a novel beadsedimentation system to count CD+ cells []. Te reagentsinclude high-density anti-hCD particles as well as anti-

hCD magnetic beads. Te device uses sedimentation o high-density, bead-associated CD+ cells as a measurementprinciple. Te height o the sedimented and visible bead/cellcolumn in a capillaryis directly proportionalto thenumber o CD+ cells when viewed against a precalibrated scale, muchlike reading a thermometer [].

Tere is no technology currently in widespread use orcounting CD at the POC.Most o the technologies describedabove have incorporated the basic tenets or POC in termso instrument size, specimen volume, reagent stability, inde-pendent power supply, and simple user interace. However,otheractors suchas throughput, associated consumables andreagents, user training, robustness o design, and the method

o analysis vary considerably.Tree assays using either scaled ow cytometric methods

(PointCareNOW and the CyFlow miniPOC) or novel tech-nologies (the Alere Pima) are also in use. Flow-based systemshave the advantage o established technology and have beenthoroughlyevaluated in larger scale ormats. Tus, evaluationo the new instruments will ocus on improving and demon-strating the robustness and the ease o use in RLS. Te needor appropriate quality controls (QC) and POC testing is anarea where QC is essential to ensure that specimen prepara-tion, equipment, and reagents are unctional, or user com-pliance with the protocol. Te Pima incorporates QC indi-cators or specimen collection and reagent and equipment

unction. Te devices being developed by Daktari and mBiooffer cassette-based instrumented readers or increased accu-racy with a simple user interace and single or no movingparts. Tis simplied instrumentation is anticipated to costless than other CD instruments in addition to havingpotentially lower ailure rates and reliance on maintenance

[].

.. Viral Load esting echnologies Overview. Viral loadtesting is the method avored or monitoring HIV patientsonce they have been initiated onto AR. High levels o HIV circulating in the bloodstream indicate that the virusis actively replicating, and these levels can be used, with theaid o molecular methods, to provide important inormationregarding the risk o disease progression and to predict theoutcome o inection []. Viral load testing is complicatedby HIV diversity and certain practical challenges, includinglaboratory inrastructure and transport o samples.

HIV is classied into two HIV- and HIV- major types.

O the two types o HIV, HIV- is predominant and has beenmost responsible or the HIV pandemic that exists today [].HIV- is divided into our groups, designated by M, N, O, andP, the main group o which is group M. Within the HIV-group M, nine clades are recognized, designated by the lettersA–D, F–H, J, and K. Recombinants between different HIV-group M subtypes are designated as either circulating recom-binant orms (CRFs) i ully sequenced and ound in threeor more epidemiologically unlinked individuals or as uniquerecombinant orms (URFs) i not meeting these criteria [].Up to CRFshave beendescribedso ar [, ]. Some CRFshave recombined urther with other subtypes or CRFs givingrise to the so-called second-generation recombinants (SGRs).

Te enormous diversity ound in HIV as discussed earlierand extraordinary ability o this virus to evolve continuously pose great diagnostic challenges. Te high level o geneticheterogeneity o HIV- and the emergence o recombinantstrains o the virus complicate viral load assay development.In an ideal world, viral load assays would detect and quantiy all known HIV- subtypes as well as inter-subtype recombi-nants and emerging variations thereon. But currently, that isnot the case, although the assays are able to recognize mostHIV- subtypes. Tereore, it is important to consider theprevalence o HIV- and HIV- groups and subtypes in aparticular geographical region when choosing a viral loadassay.

.. PCR-Based Diagnostics. In response to the HIV/AIDScrisis, access to antiretroviral therapy (AR) has increaseddramatically over the past decade in low- and middle-incomecountries []. However, successul management o HIVrequires that patients receiving AR be monitored routinely to assess treatment efficacy and detect treatment ailure dueto drug resistance and other causes. Unortunately, currentlaboratory-based methods to monitor AR are unaffordable,unavailable, or inappropriate or low-resource settings [].

Rapid antibody tests are widely available in developingnations, but they cannot be used to monitor HIV progressionor treatment efficacy. Te standard o care to monitor AR


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is quantitative viral load testing based on plasma HIV RNAconcentration []. Although CD count has also been usedto monitor AR, recent studies suggest that it may not detectearly treatment ailure adequately []. Te gold standardmethod or viral load testing, R-qPCR, is unsuitable orsettings where trained technicians, expensive reagents, elec-

trically powered equipment, and dedicated laboratory spaceare ofen unavailable. Tereore, a viral load test that isappropriate or such settings is needed.

Te NAs have the potential to offer many advantages atthe POC, such as low limits o detection and quanticationo the level o inection. PCR is an enzyme-driven processor ampliying short regions o DNA in vitro. It can createmillions o DNA copies by cycling between different tem-peratures to allow repeating steps (denaturation, annealing,and elongation) o DNA replication to take place. Despitethe simplicity and amplication power o PCR chemistry,limitations in its supporting hardware still hinder PCR romreaching its ull potential. In particular, improvements inthermal cycling speed, instrument size, and reaction volumeare still much needed. Te bulky instrumentation and largereaction volume required in conventional bench-top thermalcyclers lead to large thermal mass which reduces the temper-ature transition speed and reaction efficiency.

Rapid tests are widely used to screen or HIV inec-tion at POC and this has signicantly expanded diagnosticcapabilities o testing sites in developed countries, as wellas resource-limited settings. Despite advances made by thewidespread availability o rapid tests, all antibody-baseddetection o HIV exhibits some limitations. HIV-specicantibody typically begins to appear around three weeks aferinection, allowing or detection by most antibody-basedassays within – weeks []. Tis window period prior toor during early sero conversion may lead to alse-negativetest results in recently inected individuals. Additionally,accurate diagnosis o inants born to HIV-inected motherscanbe challenging i based solely on antibody positivity, since

vertically transerred maternal antibodies may persist or –months afer birth[]. For conrmatory diagnosiso early HIV inection or inant diagnosis, nucleic acid amplicationtests (NAA) are preerred, as HIV- RNA can be detectedas early as – days afer inection and HIV- DNA and/orRNA are denitive indicators o active inection []. Intheir current orm, however, NAAs are not easible or POCtesting, because they are time consuming, expensive, andtechnically complicated.

5. Current Isothermal AmplificationTechnologies

.. Isothermal Amplication. Isothermal amplication tech-niques operate at a single temperature, eliminating theneed or a thermocycler, enabling them to be conductedon simple and portable heating systems. Several isothermalamplication techniques have been developed in recentyears, such as helicase-dependent amplication, rolling circleamplication, and nicking enzyme amplication reaction[–]. Loop-mediated isothermal amplication (LAMP)

[] has been optimized or the detection o DNA and/orRNA (R-LAMP) rom a wide range o bacterial and viralpathogens including HIV [–].

LAMP or R-LAMP exhibits several characteristics thatare ideal or integration into a rapid nucleic acid-based diag-nostic test. Te amplication reaction requires six primers

specic or eight separate regions within the target sequence,contributing to the high specicity o the amplicationmethod. Amplied material can typically be detected within– minutes when incubated at a constant reaction tem-perature o –∘C []. LAMP has also proven to beless sensitive to biological inhibitors than PCR [], whichenables direct amplication rom clinical specimens, thereby eliminating the need or an additional nucleic acid extractionstep. Direct amplication rom plasma, whole blood, and oraluid haspreviously beendemonstrated orHIV- [, , ].Lastly, immediate visual detection o amplied products isacilitated by the large amount o DNA that is generated by each reaction. Several groups have incorporated uorescentdetection methods into the LAMP assay or real-time orimmediate naked-eye detection [, , ].

.. Nucleic Acid Sequence-Based Amplication (NASBA).Te NASBA techniques like transcription mediated ampli-cation (MA) and sel-sustained sequence replication (SR)mimic in vivo retroviral replication mechanisms to produceRNA amplicons rom an RNA template. Modied cDNAis ormed rom an RNA template, which is then rapidly amplied into RNA amplicons. Te complete process is asingle step and proceeds in a single volume with an ssRNAproduct suited or direct use with hybridization probes mak-ing NASBA very appealing or point-o-care echnologies

(POC) [].Te target RNA being labile calls or aster and careul

sampling procedures as RNA may degrade. Lower incuba-tion temperature is desirable as it will reduce the powerconsumption and thermal control complexity but may resultin a low stringency reaction environment and allow non-specic amplication, making robust primer design andassay evaluation crucial []. Furthermore, an initial ∘Cstrand separation step is required i dsDNA is to be targetedwhilst RNA amplication requires a ∘C step to removesecondary structures. Tese temperature steps will be anengineering consideration in POC devices where precisethermalcontroland associatedincreased powerconsumption

will be disadvantageous.

.. Helicase-Dependent Amplication (HDA). Te high tem-perature requirement to separate dsDNA can be overcome by using stand separating enzyme helicase in PCR amplicationo DNA. Te typical existing HDA protocol needs – min or low copy number targets. Tis shortcoming canbe overcomeby including the use o restriction endonucleasestargeting regions upstream o the target sequence to enhancehelicase activity in the target region, addition o crowdingagents, and increasing enzymeconcentrations [].Te HDAis appealing or POC as it requires single set o primersand two enzymes (three or reverse transcription-HDA), and


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is compatible or existing uorescent detection chemistries[].

... Recombinase Polymerase Amplication (RPA). RPA isa single tube, single temperature (–∘C) amplicationmethod. Te key to the amplication process is the or-

mation o a recombinase lament, a complex combining atarget-specic primer, and a recombinase enzyme. When thetarget-specic sequence is encountered by the recombinaselament, it perorms strand exchange, inserting the primeronto the target. Te displaced d-loop ormed is stabilized by ssDNA binding proteins (gp) to prevent reannealing.Spon-taneous disassembly o the recombinase lament upon strandexchange leaves the primer/target hybrid open to extensionby strand-displacing polymerase activity. Repetition o thecycle leads to geometric amplication. Favorable thermalrequirements, procedural simplicity, and very rapid ampli-cation (– min) make this recently developed process[] a leading technology or integration into POC devices.

Te added “off-temperature” ability o RPA to proceed ata variety o temperatures is o great appeal or eld applica-tions where precise temperature control is ofen technically challenging and will allow or instrument-ree amplication.Te biochemistry o RPA is incompatible with existingintercalating dyes, molecular beacons, and aqMan technol-ogy. Alternative uorescent probe detection strategies likewist-Ampexo and wistAmp have been developed to allow single tube uorescent detection using sequence-specicprobes. As an emerging technology, there is comparatively little in the published literature regarding RPA technology,RPA primer/probe design, and its integration with POCdevices.

... Loop-Mediated Isothermal Amplication (LAMP).Among the isothermal nucleic acid methods currently available, loop-mediated isothermal amplication [](LAMP) is the most widely researched and has beenwell characterized offering signicant support during thedevelopment process. LAMP is a rapid amplication methodemploying a strand displacing Bst DNA polymerase and– primers, two o which are “old back” primers []which orm stem-loops motis with sel-priming capability.Tis results in an amplication scheme where the primingsequence is copied with each round o replication andremains tethered to the previous amplicon resulting in a

concatenated product o alternating sense/antisense repeatso varied length. Subsequent studies have ound that theuse o additional “loop primers,” which bind to the loopstructures, can greatly reduce the reaction times resultingin a total o primers [].

Te –∘C reaction temperature combined with aminimum o primers makes LAMP a highly specicreaction allowing an “amplication is detection” scheme. Tisspecicity has allowed the insoluble pyrophosphate reactionby-product [] to be employed in a turbidimetric detectionstrategy or both qualitative visual indication[] or real-timequantitative turbidimetry [], which offers a very simple,robust detection strategy orPOC integration. Whilst a ∘C

initial strand separation step is not essential [], it has beenshown to increase analytical sensitivity []. As with someother isothermal methods (SMAP, NEAR, and SDA), LAMPis highly dependenton the careuldesign o multiple complexprimers and this can be overcome by the use o appropriatesofware [].

... Rolling Circle Amplication echnology (RCA). RCA isa powerul technique which exploits the strand displacementand highly processive polymerase activity o the Phi bac-teriophage DNA polymerase (QDNAP) acting on circularDNA targets [, ]. Te basic RCA reaction (linear RCAor single primer RCA) is initiated by a primer annealingto a circular ssDNA. Te QDNAP can elongate a new strand o the circular template eventually completing a loopand reaching the point o initiation. Strand displacementactivity allows the newly orming strand to continuously displace the previously generated strand as polymerizationadvances. Generation o a continuous catenated ssDNA o

up to . mega bases [] has been reported and continuesuntil an external actor, such as nucleotide depletion, haltsthe reaction. Tis continuous catenated product attached tothe template allows in situ or localized amplication, whichcanbe used to concentrate labels within a small detection areaand enumerating single DNA molecules [, , ].

... Single Primer Isothermal Amplication (SPIA). SPIA isa linear amplication technology or DNA based on repeatedreplication o target sequences enabled by the use o chimericRNA/DNA primers, which bind target regions and initiatepolymerization []. Te RNA/DNA primer is engineered insuch a way that RNase H degradation o the RNA portion o

the chimeric primer will reexpose the binding site to allow asubsequent primer to anneal. Strand displacement activity o the polymerase removes the previously generated strand. Tisrepeated cycle continuously generates new amplicons untilreagents or primers are depleted. A similar method, Ribo-SPIA, developed to ampliy total mRNA, replicates only theoriginal transcripts and not copies, resulting in a high-delity product. SPIA will become more widespread as genomicanalysis enters the point-o-care domain.

... Smart Amplication Process Version (SMAP/Smart- Amp). Tis nascent amplication technology (not to beconused with the signal amplication method SMAR,

signal-mediated amplication o RNA technology) employssimilar enzymes and sel-priming loop motis to LAMP.In contrast to the symmetrical primers o LAMP, SMAPprimers are designed asymmetrically with different tailmotis in the two target anking primers. Tis serves toreduce the ormation o background products rom misam-plication. Te amplication process occurs in two steps:an initial “key intermediate” step orming a target sequenceanked and with old-back domains to provide sel-priming ability and a second amplication step where the key intermediates undergo repeated sel-priming and rapid targetamplication resulting in concatenated, primer inclusiveamplication products.


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In addition to this amplication ormat, SMAP employsbackground suppression technology to increase specicity and permit an “amplication is detection” assay. Ultrahighspecicity is achieved by employing Termus aquaticus MutSto identiy mismatched primer/target hybrids. MutS scansdsDNA and will irreversibly bind to any mismatch duplex

with single nucleotide sensitivity []. Bound MutS preventspolymerization thus checking amplication o nonspecicsequences resulting in complete inhibition o nonspecicamplication. Te incorporation o MutS and asymmet-ric primer design permits single nucleotide discrimination,making SMAP particularly useul or SNP identication.Whilst SMAP has been shown to proceed slower thanLAMP, the SMAP technique’s very high specicity, highsensitivity ( copies), and powerul amplication (-oldlarger than PCR) combined with the developers (DNAFORMand RIKEN, Yokohama, Japan) reporting o specic detectionrom crude cell lysate make this a promising development andpotentially powerul tool or POC devices [, ].

... Strand Displacement Amplication (SDA). Tismethod relies on biunctional primers incorporating bothtarget recognition and endonucleases regions [, ].Following strand separation, these biunctional primersextend incorporating the restriction target into the amplicon.Bumper primers, which bind and extend upstream, releasethis amplicon. Successive rounds o primer binding generatedsDNA incorporating restriction sites, which can then beacted upon by the restriction endonucleases to nick a singlestrand o the newly ormed duplex. Tis nicking allows thepolymerase to displace the existing strand and incorporatea new amplicon. Tis nick and run scheme is repeated

to effect exponential amplication. Single strand nickingis effected by the incorporation o a modied adeninenucleotide, dAP aS (-O-l-thiotriphosphate), which isresistant to the endonuclease activity. Tus only the newly incorporated primer will be nicked leaving the ampliedstrand to repeatedly act as a template or primer binding. Tecomplex asynchronous reactions occur concurrently anduser interventions are limited to an initial heat denaturationwith primers ollowed by addition o polymerase andrestriction enzymes at a uC incubation, a protocolwhich is by no means complex and appears amenable toPOC use [].

... Nicking and Extension Amplication Reaction (NEAR).NEAR is a recent development o the earlier describedEXPAR reaction []. Capitalizing on nicking enzymes toexpose binding sites or primers, the EXPAR displays excel-

lent reaction kinetics and 6- to 9-old amplication ina ew minutes. However, EXPAR is limited to amplicationo sequences adjacent to native nicking enzyme recognitionsites within the target genome []. NEAR is a renemento EXPAR to allow amplication o any target by insertingnicking-enzyme recognition sites adjacent to target regions.Te two-stage NEAR reaction proceeds in a similar mannerto the SDA reaction exploiting nicking-enzymes to generatea site rom which polymerase elongation can initiate. In

contrast to SDA, the nicking enzyme employed in NEAR willonly nick a single side o a duplex, removing the need, asseen in SDA, or strand modication o the duplex to preventdouble stranded cleavage.

... Isothermal and Chimeric Primer-Initiated Amplicationof Nucleic Acids (ICAN). ICAN is a simple scheme orDNA amplication at ∘C using relatively ew reagents:a pair o -DNA-RNA- chimeric primers, thermostableRNase H, and a strand-displacing DNA polymerase [].Following initial heat denaturation o the target dsDNA,the chimeric primer binds to the template and is elongatedby BcaBES DNA polymerase. Te newly ormed strandis nicked by thermostable RNase H action, not at the

border o the chimeric primer as initially thought but at thepenultimate RNA residue, allowing the strand displacingDNA polymerase to release a newly synthesized strand witha single RNA residue, leaving the template with a truncatedprimer, which is still sufficient to prime elongation. Te cyclerepeats until the chimeric primer is sufficiently shortened,allowing a new, ree chimeric primer to anneal preerentially,recommencing the cycle [].

In addition to this multipriming model, a templateswitching mode o amplication has been identied [].emplate-switching amplication occurs when both orwardand reverse primer bind to the same dsDNA target andproceed to elongate toward one another, eventually switchingthe template rom using the original template to usingthe newly synthesized strand elongating rom the oppositeprimer as the template. Tis displaces both parent strands,orming a dsDNA o two daughter strands consisting o the target anked by the primer regions with incorporatedchimeric primer on one strand. Tis dsDNA o daughterstrands is acted upon by RNase H, which introduces a nick inthe RNA region and polymerase elongation can commence.I both orwardand reverse elongation reactions occur simul-taneously, the template switching cycle will recommencewith the parent strands being displaced and the chimericprimer-bound strands becoming the dsDNA product. I the nicking and elongation occur asynchronously, a single-stranded product is ormed, still having an incorporatedchimeric primer. Tis single strand with primer can enter themultipriming amplication cycle; thus there is no dead-endproduct and amplication will be sustained until the reagentsbecome exhausted.

... Conclusion. Many o the isothermal amplicationmethods described above display speed, amplication power,and analytical and diagnostic specicity and sensitivity equalto, and ofen in excesso, existing molecular techniques basedon real-time PCR/qPCR. Given this excellent perormanceand their suitability or miniaturization, it is highly likely that isothermal amplication strategies will become com-monplace in the next generation o point-o-care diagnosticdevices. Although tremendous progress has been made sincethe discovery o PCR, still there is no robust nucleic acid-based point o care PCR.


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6. Microfluidic Diagnostics

Tis section categorizes various uidics technologies suchas pressure-driven ows, capillary ows, electromagneti-cally driven ows, centriugal uidics, acoustically drivenows, and droplet uidics. Ten three broad categories o POC microuidic testing devices are considered: lateral ow devices, desktop and handheld POC diagnostic platorms,and emergent molecular diagnostic POC systems. Suchevolving trends as miniaturization, multiplexing, network-ing, new more sensitive detection schemes, and the impor-tance o sample processing are discussed. It is concluded thatPOC microuidic diagnostics have a potential to improvepatient treatment outcome and bring substantial savings inoverall healthcare costs.

Microuidic diagnostics had an explosive growth in thelast years spurred by the convergence o clinical diagnostictechniques (such as blood gas analysis, immunoassays, andmolecular biology testing) and mature micro abricationtechnology that allowed production o sub-millimeter-size

uidic channels and reservoirs in a variety o material sys-tems (e.g., silicon, polydimethylsiloxane (PDMS), and poly (methyl methacrylate) (PMMA)) [].

.. Microuidic echnologies. Microuidic diagnostics usetechnologies to accomplish a predetermined set o operations(i.e., to bring the sample and reagents together, to add buffer,to implement wash, and to acilitate the readout) requiredby the specic biochemistry o the tests and detection tech-niques. Tey can be classied according to uid propulsionsuch as pressure-driven ow and electromagnetically drivenow. Tey can also be classied according to the type o

ow like continuous ow or the so-called segmented ow (where uid is advanced in discrete packets or droplets).Te segmented ows (also called droplet microuidics), anextremely important emerging technology, can be achievedon a variety o platorms, or example, centriugal platorms,pressure driven platorms, and electromagnetically drivenows.

Strip-based tests that use capillary orces are among themost ubiquitous and commercially successul POC tests.Tere are many evolving LF testing technologies like nucleicacid hybridization-based LF devices [] or a combination o antibody-antigen recognition with nucleic acid hybridizationin nucleic acid lateral ow immunoassays [] and utilization

o advanced labels such as resonance-enhanced absorp-tion [], chemiluminescence [], upconverting phosphors[], silver-enhanced gold nanoparticle labels []. Othertrends involve developing a larger number o immunoassays[], advancing quantication o the detection [], andquality control in manuacturing to increase reproducibility o tests.

.. A Lateral Flow Assay for Quantitative Detection of Amplied HIV- RNA. Rebecca R. Richards-Kortum’s grouprom Rice University, Houston, X, have described a lat-eral ow assay that employs gold nanoparticle probes andgold enhancement solution to detect amplied HIV RNA

quantitatively. Tey showed, coupled with nucleic acidsequence-based amplication (NASBA), that this assay candetect concentrations o HIV RNA that match the clinically relevant range o viral loads ound in HIV patients. Teirlateral ow assay achieved a resolution o . log

10 copies/mL

over a linear range that extends . orders o magnitude

ranging rom . to log10 RNA copies. When coupled withNASBA, the LFA can detect copies o HIV gag RNA. Wespeculate that this LOD corresponds to a plasma viral loado roughly copies/mL, assuming that a plasma sample

volume o mL will be used and that hal o viral RNA romthe sample is added to the NASBA reaction. Te perormanceo the LFA when detecting NASBA products suggests that theLFA may be sufficient to detect signicant changes in viralload, suppression o viral replication, and therapeutic ailure.Te LFA only requires a heat block, scanner or camera, andpipette. Te lateral ow assay uses a sample volume o mL,requires only three steps over the course o minutes, costs. per strip using commercial reagents, and perormsconsistently afer short-term storage. By modiying the targetcapture, positive control, and probe sequences, the LFA may be adapted to detect other RNA targets. Te LFA is capableo detecting short, amplied sequences or long, genomicsequences. Te LFA is suitable or low-resource settings andhas the potential to be perormed at the POC [].

.. Miniaturization of NA Instrumentation. Miniaturiza-tion o NA instrumentation has been the ocus in recenttimes; Genie II (OptiGene Horsham, UK, and wista(wistDx, Cambridge, UK) demonstrates the possibility ormore portable NA equipment. []. Separate amplicationand detection o the PCR product allow or greater sensitivity

and elimination o nonspecic signals. With regard to POCapplications, the requirement o multiple procedural stepsor amplication and separate detection is cumbersome.However, development o simplied assays is limited by theneed to maintain analytical sensitivity and specicity in aprotocol consisting o ewer steps.

.. Combined Amplication and Detection. Te advent o uorescent DNA probes and intercalating dyes [, ] hasallowed the real-time quantication o amplication productsin both PCR and isothermal amplication reactions. Telimiting actor o such systems is the need to maintainanalytical sensitivity to the target whilst developing reaction

conditions with sufficiently high analytical specicity to elim-inate nonspecic products accumulating and producing aalse-positive result. Te use o sequence-specic uorescentprobes such as aqMan and molecular beacons offers thebenets o combined amplication and detection in reactionslacking the specicity or nonspecic detection methods.Probe-based real-time detections also allow or multiplexingusing different probe uorophores. Te simplicity o com-bined amplication and detection in a single step is desirableand will hasten the time to result.

.. Amplication-Free Direct Nucleic Acid Detection. Suchtechniques use highly sensitive detection strategies to identiy


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target sequences in a sample without the need or nucleic acidamplication. Tis offers the potential or simplied proto-cols, reduced reagent consumption, and simplied operatingplatorms. Te success o such systems is entirely dependenton the development o robust biosensors with analyticalsensitivity sufficient to detect the very low abundance NA

in complex clinical samples whilst maintaining appropriateclinical specicity or diagnostic use. Non-POC systemsincorporating direct NA detection are commercially avail-able. For example, NanoString (Seattle, USA) offers a bench-top instrument using molecular barcodes or gene analysis.Portable systems or direct detection, such as those beingdeveloped by Geneuidics (Irwindale, USA) using electro-chemical detection and ExoCyte (Reading, UK) employingsilicon nanowire, carbon nanotube, and quantum dot tech-nologies, have yet to be cleared or diagnostic use. Directdetection technologies are expected to enter the global testingmarket within the next years and have the potential todisplace numerous methods currently in use [].

7. Paper-Based Sensors

Paper is an extremely versatile material possessing desirableproperties such as mechanical exibility and strength. Temain constituent o paper is cellulose ber, and this can behighly attractive or certain applications as it allows liquidto penetrate within its hydrophilic ber matrix without theneed o an active pump or external source []. More-over, cellulose bers can be unctionalized, thus changingproperties such as hydrophilicity, i desired, as well as itspermeability and reactivity. It can be produced sustainably and inexpensively, is ubiquitous globally, and provides anexcellent substrate or chemical ctionalization, making itwell suited to use in low-cost, point-o-care (POC) diagnosticdevices. In recent times, paper has been employed in theconstruction o more complicated analytical devices knownas lab-on-a-chip (LOC). Conventional LOC devices ofenmade o glass or PDMS, while considerably more simpliedthan their ull-scale laboratory counterparts, still maintain acost which is prohibitive to their deployment in economically developing regions due to the specialist equipment and cleanrooms required or their manuacture []. In , the rstpaper device or the semiquantitative detection o glucosein urine was demonstrated [], that urther developed intoimmune chromatographic paper test strips (also known as

lateral owor dipstick tests), with the pregnancy test kit beinga well-known example []. Paper has been suitably modi-ed to detect analytes by different methods like colorimetric,electrochemical, electrical conductivity, chemiluminescence,and electro-chemiluminescence [].

Paper-based sensors are a new alternative technology orabricating simple, low-cost, portable, and disposable analyti-cal devices or many application areas including clinical diag-nosis, ood quality control, and environmental monitoring.Te unique properties o paper which allow passive liquidtransport and compatibility with chemicals/biochemicals arethe main advantages o using paper as a sensing platorm.Depending on the main goal to be achieved in paper-based

sensors, the abrication methods and the analysis techniquescan be tuned to ulll the needs o the end user. Currentpaper-based sensors are ocused on microuidic delivery o solution to the detection site whereas more advanceddesigns involve complex D geometries based on the samemicrouidic principles. Although paper-based sensors are

very promising, they still suffer rom certain limitations suchas accuracy and sensitivity. However, it is anticipated that inthe uture, with advances in abrication and analytical tech-niques, there will be more new and innovative developmentsin paper-based sensors. Tese sensors could better meet thecurrent objectives o a viable low-cost and portable devicein addition to offering high sensitivity and selectivity andmultiple analyte discrimination.

.. CMOS Cell Sensors for Point-of-Care Diagnostics. Com-plementary metal-oxide-semiconductors- (CMOS) basedproducts can enable clinical tests in a ast, simple, sae,and reliable manner, with improved sensitivities. Extensive

use o CMOS-based sensors in DNA diagnostics [, ]and commercialization o CMOS-based DNA sequencingsystems (Ion orrent Systems) [] is already promising orthe involvement o CMOS-based cell detection systems inmedical applications.

.. Challenges in Paper Platforms. Although there is enor-mous potential in paper as a platorm or LOC devices, thereis a lot o scope to improve in terms o sensitivity. Tis istypically due to how the sample is introduced onto the paper,which can be at a considerable distance o to millimetersrom the detection zone. As the analyte is delivered across thepaper, the local analyte concentration may decrease as a result

o solution spreading and may also evaporate i the distanceand time o travel are ar rom the point o introduction. Teother problems are encountered in multiplex analysis as thereis the potential or cross-talk as some o the signal reportersmay diffuse to neighboring channels []. Tere are limitedchemistries available or the conjugation o biomolecules tocellulose or use in biomedical applications.

.. HIV Subtypes Isolation on Chip. Utkan Demirci grouphave demonstrated a microuidic chip device that can effec-tively capture various subtypes o HIV particles throughanti-gp antibodies, which were immobilized on themicrochannel surace. Tey showed that Protein G-based

surace chemistry has a better control over the antibody orientation on the surace compared to antibody immo-bilization methods including passive adsorption, covalentbinding, and NeutrAvidin-based surace chemistry. Tisplatorm technology can be potentially used to measure HIV- viral load in resource-constrained settings. Protein G-basedsurace chemistry when used together with NeutrAvidin-based surace chemistry enables the separation o captureand detection chemistries that can potentially reduce thenonspecic binding and enhance detection outcomes Teirimmunosensing device enables the development o POC on-chip technologies to monitor viral load and guide antiretro-

viral treatment (AR) in resource-constrained settings [].


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8. Fraunhofer ivD-Platform

A consortium o seven Fraunhoer Institutes have developeda lab-on-chip system called “Fraunhoer ivD-platorm” whichcan be potentially used as POC diagnostics. Te platorm ea-turesa highdegree o modularity andintegration. Modularity allows the adaption o common and established assay typeso various ormats. Integration lets the system move romthe laboratory to the point-o-care settings with multiplexingcapability. By making use o the microarray ormat, the lab-on-chip system also addresses new trends in biomedicine.Te low-cost device has reagents reservoirs, microuidicactuators, and various sensors integrated withinthe cartridge.In combination with ully automated instrumentation (read-out and processing unit), a diagnostic assay can be perormedin about minutes. Tis is possible with user-riendly inter-acing read-out unit, data acquisition, and data analysis unitstogether.

Te assays or nucleic acids (detection o differentpathogens) and protein markers (such as CRP and PSA)

have been done using an electrochemical sensor based onredox cycling or an optical sensor based on total internalreectance uorescence (IRF). Furthermore, integration o sample preparation and polymerase chain reaction (PCR)on-chip has been done. Te instrument is capable o providingheating-and-cooling cycles necessary or DNA amplication[].

9. Detection

Electrochemical and optical technologies are the clear leadersin detection technologies in the market as they do not use

complicated instrumentation or detection. Tereore point-o-care detection should be able to detect signal withoutusing any complicated instrumentation or i the results canbe visualized.

.. Optical Detection. Optical detection is the simplest andmost popular method used in immunoassay applications.Most immunosensors are based on optical detection andcommonly use a label (e.g., a uorescent label, enzyme, ormetallic particle) that may acilitate optical signal enhance-ment and increase detection sensitivity. Optical immunosen-sors combined with microuidic chips have recently beenproposed as an attractive immunosensing platorm, and

many reviews have explored their potential applications inclinical diagnostics, particularly because there is a growingneed to simultaneously screen multiple proteins in a singlesample. Te optical detection method, which can be easily implemented in microuidic systems, is a prime candidateor this multiplexed analysis. Optical detection methods canbe divided into ve main categories on the basis o detec-tion signal: uorescence, luminescence, absorbance (col-orimetry), surace plasmon resonance (SPR), and surace-enhanced Raman scattering (SERS); each technique hasinherent advantages and disadvantages. In this section, weocus on the recent multiplexing applications in microuidicimmunosensors using optical detection techniques.

Tere are several optical detection methods used or POCapplications suchas uorescence with variants suchas Forsterresonance energy transer (FRE) and upconverting phos-phor technology, luminescence, absorbance (colorimetry),surace-plasmon resonance (SPR), and various categorieso light scattering: Rayleigh (particles much smaller than

wavelength), Mie (particles comparable to wavelength, shapedependent), geometric (particles larger than wavelength),resonant (wavelength overlaps an electronic transition o the particle), and Raman (vibrational quanta added to orsubtracted rom the excitation wavelength) []. Te mostcommonly used technique is absorbance as it is commonly used in LFAs based on gold or polymer (nano-) particles.However uorescence is used or the broadest range o different types o POC assays [] or reasons o sensitivity and, more recently, the ready availability o a range o different colors o efficient uorophores, including quantumdots, quantum-dot barcodes, and uorescent nanoparticles,providing improved limits o detection. Fluorescence in somecases can detect single particle LODs enabling multitargetmultiplexing [].

Te supercritical angle uorescence (SAF) which is beingused recently detects uorescence emitted in close proxim-ity to a uorophore-supporting optically transparent chipsurace. Tis method provides substantial enhancement o uorescence collection efficiency while rejecting backgroundrom unbound uors or impurities, as it connes the u-orescence detection volume to material within about onewavelength o the chip surace [].

Generally the analyte is labeled by attaching a chro-mophore, uorophore, or particle (dye containing, semi-conductor/quantum dot, noble metal, or scattering) to anantibody or nucleic acid strand that coners specic recogni-tion or optical detection. Nanoparticles including quantumdots are nding increasing application. Nanoparticle labelsconjugated with biomolecules have been used in a variety o different assay application. Nanoparticles offer adjustableand expandable reactive surace area compared to the moretraditional solid phase orms utilized in bioaffinity assays dueto the high surace-to-volume ratio. Signal enhancement by conjugating nanoparticles with uorescent, luminescent, andother measurable properties has enhanced detection limit by several olds. Te potential to multiplex along with the ability to increase sensitivity and specicity without using enzymeshas increased the use o nanoparticles in immunoassays orearly detection. It has been shown that using time resolved

uorescence o lanthanides like europium nanoparticles withlong stokes shif can reduce background and increase detec-tion limit to as low as . pg/mL o HIV p []. Te deter-mination o cancer biomarkers in serum and saliva usingquantum dot bioconjugate labels are used. Quantum dotswere employed on chip or CD+ -cell counting in a POCapplication. Aptamers were tethered to gold nanoparticles aspart o an LFA-like dry-reagent assay strip to detect thrombin[]. Termal-lens microscopy (LM), an alternative touorescence detection, also uses dye labeling or detection.LM detection integrated to a miniaturized ELISA devicealong with optical, electronic, and uidic components coulddetect an LOD o ng/mL or total IgE measurement [].


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.. Electrochemical Detection

... Electrochemical Immunosensors. Electrochemical im-munoassays are the most commonly used analytical tech-niques or the quantitative detection o biomolecules, ol-lowed by optical methods. Electrochemical immunosensorsare not only sufficient to meet the demands or sensitive,rapid, and selective determination o analytes but can also beincorporated into robust, portable, and miniaturized devices.Specically, the integration o electrochemical detection withmicrouidic chips offers an attractive immunoassay platormwith signicant advantages derived rom the combinationo electrochemical analysis and a microuidic system [].In broad terms, electrochemical immunosensors unctionby detecting an electrical signal that arises rom specicimmunoreactions that occur at the surace o an electrode.According to the type o electrical detection signal, electro-chemical techniques are classied into three basic categories:

voltammetry (current), potentiometry (potential shif), andimpedimetry (resistance) [].

Some analytes are electroactive and can be measureddirectly without labeling; electrochemical detection ofen uti-lizes tagging oranalyte specicity with either an electroactivespecies or an enzyme that converts an electrochemically silentspecies into electroactive one; this approach also providessignal amplication o multiple orders o magnitude, withdetection limits below pM readily accessible [, ]. Ithas been shown that ultrasensitive capacitive immunosensoris capable o detecting subattogram per milliliter concentra-tions o p antigen [].

... Piezoelectric Immunosensors. Piezoelectric devices

convert a physical or mechanical change into electricalenergy and vice versa. Te commonest piezoelectric sensoris the quartz crystal microbalance (QCM), which exploitsthe change in the resonance o quartz crystals upon changesin their mass, allowing binding o antigen to antibody (whenone o these is immobilised on the crystal surace) to bemeasured electrically [].

... Microcantilevers (MC). Sensors based on MCs arean attractive method or sensitive and label-ree detection.On the basis o advances in micro abrication techniques,massively parallel cantilevers can be acilitated in microuidicdevices to independently monitor multiple targets []. wo

distinct modes o MC immunosensors that are utilized orthesignaltransduction o immune recognition binding events onthe MC surace are the deection (cantilever bending) andresonance (resonant requency shif) modes.

Microcantilever-based devices were developed utilizingrotating resonance microcantilevers which measured therequency-shif o the microcantilever motion with respect tothe specic adsorbed mass, to give sensors capable o mea-suring -etoprotein to less than ng mL or the label-ree,POC or early detection o hepatocellular carcinoma [].Also a microring resonator immunosensor has been recently described which can detect multiple analytes (PSA, -etoprotein CEA, tumor necrosis actor- and interleukin-)

concurrently, without loss o sensitivity or measurementprecision when compared to single-parameter analysis [].

.. Magnetic Detection. Magnetic particles are widely usedor POC diagnostics because they can be used to precon-centrate and they can also be used as a labeling technology

or detection without the requirements o uors or opticaltransparency. Magnetic particle detection technology hasevolved rapidly, the most promising and sensitive methodsnow using the giant magnetoresistance (GMR) effect withdetectors based on the so-called spin valve (SV) or magnetictunnel junction (MJ) methods [].

... Label-Free Methods. Te label-ree methods or POCdetection include SPR, amperometric immunosensor, andelectrochemical sensors based on mechanical transductionand direct electrochemical and optical transduction or ana-lytes possessing suitable characteristics.

Silicon nanowire (SiNW) biosensors have been exten-

sively studied in the last decade or the detection o biologicalmolecules as highly sensitive, label-ree, and electrical tools.SiNW biosensors hold great promise to realize POC devicesor disease diagnostics with potential or miniaturization andintegration [].

Optical surace plasmon resonance (SPR) biosensorsrepresent the most advanced and developed optical label-reebiosensor technology. Optical SPR biosensors are a poweruldetection and analysis tool that have vast applications ininectious disease diagnostics [].

Mechanical transducers or POC applications oscillateor resonate, including micro- and nanocantilevers [], aswell as various acoustic wave devices such as the quartz-

crystal microbalance (QCM) and a range o devices in thesurace acoustic wave amily []. Operating characteristicssuch as requency and signal attenuation or piezoelec-tric devices or resistance and amplitude or piezoresistive(silicon) devices are affected by the mass and mechanicalproperties o molecules and materials linked to their oscil-lating suraces, like SPR; they require only an immobilizedselective recognition layer. Nonetheless, “mass tags,” denseparticles (typically Au) that bind selectively to the target, cansignicantly enhance sensitivity. Te “bond rupture sensors,”use acoustic energy to rupture bonds between immobilizedcapture antibodies and target microbes. Because the energy and requency o added energy can be adjusted, nonspeci-

ically and specically captured particles can be desorbeddifferentially according to the bond strengths and masses,providing a unique discrimination mechanism [].

... Integrated Rapid-Diagnostic-est Reader Platform ona Cell Phone. Aydogan Ozcan group have demonstrated acell phone-based rapid-diagnostic-test (RD) reader plat-orm that can work with various lateral ow immuno-chromatographic assays and similar tests to sense the pres-ence o a target analyte in a sample. Tis compact and cost-effective digital RDreader, weighing only g, mechanically attaches to the existing camera unit o a cell phone, where

various types o RDs can be inserted to be imaged in


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reection or transmission modes under light-emitting diode-(LED-) based illumination. Captured raw images o thesetests are then digitally processed (within less than . secondsper image) through a smart application running on the cellphone or validation o the RD, as well as or automatedreading o its diagnostic result. Te same smart application

then transmits the resulting data, together with the RDimages and other related inormation (e.g., demographicdata), to a central server, which presents the diagnosticresults on a world map through geotagging. Tis dynamicspatiotemporal map o various RD results can then be

viewed and shared using internet browsers or through thesame cell phone application. Tey have tested this platormon malaria, tuberculosis (B), and HIV RDs by installing iton both Android-based smart phones and an iPhone [].

... Plasmonic ELISA. Molly M. Steven’s group havedemonstrated an ultrasensitive ELISA, which uses a signal-generation mechanism or biosensing that enables the detec-

tion o a ew molecules o analyte with the naked eye.Te enzyme label o an ELISA controls the growth o goldnanoparticles and generates colored solutions with distincttonality when the analyte is present. HIV- capsid antigen pwas detected in whole serum at the ultralow concentrationo ×−18 g/mL o p and was also detected with the nakedeye in the sera o HIV-inected patients, showing viral loadsundetectable by a gold standard nucleic acid-based test [].

10. Opportunities and Challenges forCost-Efficient Implementation of New Point-of-Care Diagnostics for HIV

High-quality diagnostics are essential to ght against HIVand to reduce its transmission. Tere are clear diagnosticneeds where conventional laboratory support is insufficientand not cost effective. HIV rapid point-o-care (POC) testingor initial HIV diagnosis has been successul, but severalneeds remain. Despite its clear advantages, POC testinghas important limitations, and laboratory-based testing willcontinue to be an important component o uture diagnosticnetworks [].

Many middle- and low-incomecountriesimplementationo rapid, cost-effective, POC HIV diagnostic screening havedeveloped algorithms or diagnosing HIV inection usingstrategic orthogonal testing assays without the need or addi-

tional conrmatory testing []. Use o saliva has broadenedthe testing but saliva samples have been associated withhigher alse-positive rates in some testing sites []. Despitethe advances and successes o HIV serological rapid assays,unmet gaps or qualitative POC applications remain. First,we need to identiy acute HIV inection because recently inected individuals are seronegative but have extremely high viral loads, making them ar more inectious comparedwith chronically inected individuals []. Early detectioncan result in early treatment initiation, which limits diseaseprogression and reduces transmission [].

Second, we need to diagnose inantsborn to HIV-inectedmothers, particularly in sub-Saharan Arica. Due to the

placental transer o antibodies during pregnancy, inantsmay test positive on serological assays yet may not be inectedwith HIV []. Currently, inant diagnosis is primarily perormed by collecting dried blood spots and transportingthe samples to central testing laboratories or DNA testing[]. However, many inants go undiagnosed because o

long turnaround times or insufficient inrastructure (e.g.,unreliable sample transportation) [].Tird, we need appropriate tests or individuals vac-

cinated with experimental HIV antigens in clinical tri-als [, ]. Vaccine volunteers mount specic immuneresponses to the vaccine constructs, which react with many serological diagnostic tests, making uture HIV diagnosisdifficult, potentially unblinding trial staff, and negatively impacting society []. Similar assays may also be benecialin resource-limited settings to monitor antiretroviral therapy (AR) effectiveness and the potential need to switch therapy []. In each o these cases, new rapid molecular POC HIVscreening tests will ll an important diagnostic gap.

11. Future Trends

Strip-based tests with the use o low-cost polymer substrates,like paper and thread microuidics, and low-cost readerslike smart phones are also expected to acilitate home test-ing. Digital/droplet microuidic devices are being actively researched and only recently have begun to enter the eldo POC diagnostics will become popular. Te gap between“traditional” (e.g., porous nitrocellulose) lateral ow assay devices and microuidic platorms is narrowing with thedevelopment o these technologies. While PCR remains agold standard or high sensitivity and specicity in mostcases, there are other emerging related isothermal PCR

techniques to consider. Tere are a range o related PCR techniques such as RCA, LAMP, NASBA, MDA, MA,SDA, and LCA (rolling-circle amplication, loop-mediatedisothermal amplication, nucleic acid sequence-based ampli-cation, multiple-displacement amplication, transcription-mediated amplication, strand-displacement amplication,and ligase chain reaction, resp.), as well as cleavase Invaderwhich have the potential to be used in POC settings. Impor-tantly, many o these methods are isothermal and operateat lower temperatures than PCR, making their integrationwith microuidic technologies more straightorward. Fordetection, new optical methods exploit phenomena includingupconversion, high-brightness nanoparticles, total-internal-

reection uorescence (IRF), SAF, FRE, and a range o plasmon-based effects. In many cases, nanoparticles enhanceoptical detection. Te abo

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Point of Care Technologies for HIV