Review Article Point of Care Technologies for HIVdownloads.hindawi.com/journals/art/2014/497046.pdf · 2019. 7. 31. · ongoing monitoring of HIV. Among those that present the most

Review ArticlePoint of Care Technologies for HIV

Mohan Kumar Haleyur Giri Setty and Indira K Hewlett

Laboratory of Molecular Virology Division of Emerging Transfusion Transmitted DiseasesCenter for Biologics Evaluation and Research Food and Drug Administration Bethesda MD 20892 USA

Correspondence should be addressed to Mohan Kumar Haleyur Giri Setty mohanhaleyurgirisettyfdahhsgov

Received 17 April 2013 Accepted 18 November 2013 Published 21 January 2014

Academic Editor Bryce D Smith

Copyright copy 2014 M K Haleyur Giri Setty and I K Hewlett This 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 of HIVAIDS requires early diagnosis initiation of therapy and regular plasma viral load monitoring of theinfected individual In addition incidence estimation using accurate and sensitive assays is needed to facilitate HIV preventionefforts in the public health settingTherefore more affordable and accessible point-of-care (POC) technologies capable of providingearly diagnosis HIV viral load measurements and CD4 counts in settings where HIV is most prevalent are needed to enableappropriate intervention strategies and ultimately stop transmission of the virus within these populations to achieve the future goalof an AIDS-free generation This review discusses the available and emerging POC technologies for future application to theseunmet public health needs

1 Introduction

Acquired immunodeficiency syndrome (AIDS) is a diseaseof the human immune system caused by the HIV [1] whichdestroys CD4+ T lymphocytes of the immune system thatprevent infections AIDS is one of the most serious globalhealth problems of unprecedented dimensions and is oneof the greatest modern pandemics At the end of 2010 anestimated 34 million people were living with HIV globallyincluding 34 million children under 15 years of age Therewere 27 million new HIV infections in 2010 including390 000 among children less than 15 years [2]

The annual number of people newly infected with theHIV has declined 20 from the global epidemic peak in1998 mainly due to tremendous progress in diagnosticsand treatment Several diagnostic technologies have emergedwith high specificity sensitivity and accuracy to detect HIVinfectionHIV testing plays an important role inHIV preven-tion in that knowledge of HIV status has both individual andpublic health benefits Early and accurate detection of HIVinfection is important to public health because this stage ischaracterized by high infectiousness and transmissibility ofthe virusThe individual benefits of HIV testing are primarilyassociated with individuals accessing care and treatment

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

The past decades have witnessed enormous technologicalimprovements towards the development of simple cost-effective and accurate rapid diagnostic tests for detectionand identification of infectious pathogens There is growingdemand within the global health community to find waysto simplify and improve the efficiency of diagnostics forHIVAIDS without diminishing the quality of patient careAt the same time there is a need to significantly increase thelevel of access to robust high-quality diagnostics in resource-limited settings in order to facilitate HIV prevention throughearly detection and treatment Low-cost technologies todiagnose and monitor HIV infection in developing countriesare a major subject of current research and health carein the developing world With the great need to increaseaccess to affordable HIV monitoring services in rural areasof developing countries much work has been focused onthe development of point-of-care (POC) technologies thatare affordable robust easy to use portable and of sufficientquantitative accuracy to enable clinical decision making Fordiagnosis of HIV infection some low-cost tests such aslateral flow tests and enzyme-linked immunosorbent assays

Hindawi Publishing CorporationAIDS Research and TreatmentVolume 2014 Article ID 497046 20 pageshttpdxdoiorg1011552014497046

2 AIDS Research and Treatment

are already in place and well established However portablequantitative tests for rapid POC HIV monitoring have onlyrecently been introduced to the market

There is great need for initial diagnosis staging andongoing monitoring of HIV Among those that presentthe most persistent challenges to improved access andefficiency are CD4 viral load and early infant diagnosis(EID) The great majority of testing options available todayare laboratory-based platforms performed 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 for patients in periurbanand rural settings The success of high-quality antiretroviraltherapy (ART) depends on simple affordable reliable andquality-assured POC diagnostics for use in resource limitedsettings (RLS) POCdiagnostics canmakeARTmore scalableand will allow ART service delivery to be significantlydecentralized to the community level At the same timesimplifying diagnostic technologies that could potentiallyreduce the cost of diagnosing and monitoring patients livingwith HIVAIDS without diminishing the quality of care isimportant for treatment success The World Health Orga-nization (WHO) has set criteria for at or near POC usemdashknown as ldquoASSUREDrdquo criteria meaning that they are (or willbe) affordable sensitive specific user-friendly robustrapidequipment-free and deliverable to those who need the test[3]

POCdiagnostics are in vitrodiagnostics (IVD) that donotinvolve the use of laboratory staff and facilities to provide theresultThe analytical ldquotargetsrdquo include proteins nucleic acidsmetabolites drugs dissolved ions and gases human cells andmicrobes Test samples can be blood saliva urine or otherbodily fluids or (semi-) solids Whether used ldquonear patientrdquoin a hospital clinic or doctorrsquos office or administered at hometo maintain health manage disease or monitor therapy or inthe field to test the safety of water food or compliance withlaws and regulations these tests accept a sample with little orno preparation and provide a result the ldquoanswerrdquo in secondsto hours [4]

The 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 of coloron a strip of paper or polymer increasingly however readersranging from hand-held devices to bench-top instrumentsread the analytical test provide a comprehensible resultand if necessary control and operate the sample-containingplatform that executes the analytical process These devicesshould be capable of using nanoliters to milliliters of complexbiological media with femtomolar to millimolar concentra-tions of analytes [5] The devices should be inexpensivedisposable units or cartridges that include microfluidic fea-tures to provide or control sample preparation flow ratemixing with reagents reaction time associated with bindingevents filtration of nonanalytical components of the sampleseparation of interfering agents and of multiple analytes andan effective measurement capability [6]

ldquoWhy POC Diagnostics for HIVrdquo POC measurementsprovide results rapidly where needed and often with major

time savings samples do not travel to a laboratory to awaitthe attention of 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 for accuracy or reliability

2 HIV Diagnostics

Diagnostics for HIVAIDS can generally be divided intothree test categories (i) tests to facilitate initial diagnosis(ii) tests to stage the patient and (iii) tests to monitor thepatient both before and after initiation of ART There aregenerally accepted algorithms and tests used at each stage ofthe infection [7]

21 Low-Cost Diagnostic Tools for Monitoring HIV Low-costtechnologies to diagnose andmonitorHIV infection in devel-oping countries are a major subject of current research inhealth care settings in the developing world With increasingneed to provide access to affordable HIVmonitoring servicesin rural areas of developing countries much work has beenfocused on the development of point-of-care technologiesthat are affordable robust easy to use and portable and ofsufficient quantitative accuracy to enable clinical decisionmaking For diagnosis of HIV infection some low-cost testssuch as lateral flow tests and enzyme-linked immunosorbentassays are already in place and well established Howeverportable quantitative tests for rapid HIV monitoring at thepoint of care have only recently been introduced to themarket[8] The Ora Quick Rapid HIV-12 Antibody Test is a lateralflow rapid test for oral fluid specimens that perform as well asblood-based tests even at low concentrations ofHIV antigensin oral fluid [9] The Aware HIV-12 U with 972 sensitivityand 100 specificity 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 p24 antigen aswell as tests for early infant diagnosis and for HIV-1 andHIV-2 subtype differentiation [10] Detection of both antibody andantigen during the acute phase of infection is particularlybeneficial to prevention efforts because the rate of HIVtransmission is several folds higher during this phase and canaccount for up to 50 of newHIV infectionsThe DetermineHIV-12 AgAb Combo is an immunochromatographic rapidtest that detects antigen and antibody separately In twoindependent studies the Determine HIV-12 AgAb Comboshowed sensitivities of 50 and 86 for antigen detection[11 12]

22 HIV Viral Load Monitoring HIV viral load monitoringis crucial for therapy and staging of the disease Reverse tran-scriptase (RT) an enzyme present during viral replicationcorrelates linearly with HIV load and CD4+ T-cell count [13]Measuring this RT protein may be an alternative for resourcelimited settings as its yielding results in a few hours and costs80 less than nucleic acid tests [14]

AIDS Research and Treatment 3

Viral load estimates based on reverse transcriptase testsare not directly comparable to those obtained through PCRbecause they have been found to underquantify the HIVload [15 16] However in places where PCR is unavailableresults of reverse transcriptase tests can be a cheaper viralload monitoring method Further advantages of reverse tran-scriptase tests include their ability to detect multiple subtypesof HIV and their possible use in monitoring HIV load inpediatric patients [14] Levels of theHIV core protein p24 alsocorrelate with HIV load and CD4+ T-cell count Assays forp24 have been shown to have lower sensitivity and there hasbeen difficulty in correlating plasma p24 and viral RNA loadin persons receiving ART [17] However improvements insensitivity of p24 antigen tests may allow further reevaluationof these assays for HIV viral load monitoring

221 Current Viral Load Technologies HIV viral load tech-nologies can be categorized broadly as nucleic acid-basedtests (NAT) and non-NAT-based technologies NAT tech-nologies detect and quantify viral RNA whereas non-NATtechnologies detect and quantify HIV viral enzymes andproteins that can be correlated with the amount of viralRNA Most of the commercial assays use target amplifica-tion methods [3] In general the advantages of NAT-basedapproaches are their wide availability in quality-assured kitsand clinicians have familiarity with interpreting the resultsThe assays vary in terms of sample preparation and amplifica-tiondetection methodologies The major NAT-based assaysand platforms are discussed below [3]

There are four commercially available RT-PCR basedviral load assays at present (i) COBAS AMPLICOR HIV-1 MONITOR v15 (Roche Molecular Systems) (ii) COBASAmpliPrepCOBAS TaqMan v20 (Roche Molecular Sys-tems) (iii) Real-Time HIV-1 (Abbott) and (iv) VERSANTHIV-1 RNA 10 assay (kPCR) (Siemens) There are also anumber of other in-house procedures and test systems thathave good sensitivity and reproducibility and are used invarious countriesThe non-NAT-based technologies quantifyproteins and enzymes specific to HIV These include assaysthat measure the level of reverse transcriptase activity andassays that measure the concentration of circulating p24protein [3]

222 Reverse Transcriptase Technologies Reverse transcrip-tase (RT) assays detect that viral enzyme the RT activity canbe quantified and levels can be correlated with the amountof HIV Therefore an assay for RT can reflect the HIV viralload in the patientrsquos blood RT assays originally requiredradioisotopes a scintillation counter and an ultracentrifugefor performance but they have been simplified andmade lesshazardous Currently there is one RT platform available forin vitro usemdashthe ExaVir Load manufactured by Cavidi AB[3 18]

23 ExaVir Load (Cavidi AB) Cavidi manufactures theExaVir (Version 3) which is a quantitative HIV-RT test that isdesigned to measure viral-bound HIV RT activity in plasmain order to estimate the HIV viral load The ExaVir Load

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

An advantage of the assay is that because the ExaVirLoad determines viral load based on quantification of RTactivity and does not target a specific nucleic acid sequenceit can measure any HIV type or subtype with high accuracyincluding O and N groups The measuring range of the assayis the equivalent of about 200 to 600000 copiesmL (or1 to 3000 femtograms fgmL) There is performance dataavailable on the ExaVir Load showing good correlation withthe COBAS AMPLICORMonitor assay [3 18]

231 p24 Antigen Technologies HIV-1 infection is charac-terized by an early spike in HIV-1 antigens in the bloodDuring this period of acute infection or antigenemia theantigens in the blood are detectable but in most individualsthe antigen levels subsequently become undetectable for aperiod of time It is only later in HIV disease with increasingfailure of the patientrsquos immune system and an increasinglevel of the virus that the antigens may again becomedetectable in the blood One of the viral components inblood during the period of antigenemia is the core proteinp24 the major internal structural protein of HIV-1 The p24appears within 2 weeks after infection as a result of theinitial increase in viral replication and is associated withthe period of antigenemia during which the individual ishighly infectious Testing for p24 antigen can be of value inseveral circumstances (i) detecting early HIV infection (ii)diagnosing infection in infants (discussed later in this report)and (iii)monitoringART In the past before the availability ofNAT-based technologies the p24 antigen assay was used formonitoring the development of AIDS andmonitoring diseaseprogression [19] In particular the NEN HIV-1 p24 ELISAassay from Perkin Elmer (an ultrasensitive heat denaturedp24 antigen quantification assay) has been used for thispurpose However the p24 antigen tests available were notvery sensitive and there are concerns about correlation of p24with HIV RNA [20] Moreover with a linear range between10000 and 30000 RNA copy equivalentsmL the assay is oflimited utility in detecting early treatment failure and it is notuseful in patients with low viral replication [20] Thereforethe use of p24 antigen testing will not be discussed further inthis report in the context of monitoring patients on ART butwill be revisited in the discussion of EID

The latest laboratory immunoassays to come to themarket for central laboratory use are antigenantibodyimmunoassays which can detect p24 antigen in additionto detecting anti-HIV IgM and IgG Combination anti-genantibody assays have been approved and used in manycountries since the late 1990s [21ndash25]These assays detect p24antigen at the level of 11ndash18 pgmL [26] which is equivalent toapproximately 30000ndash50000 copiesmL of HIV RNA [27]Recently two such assays (Abbott ARCHITECT HIV AgAbCombo and Bio-Rad GS HIV Combo AgAb EIA) have beenapproved for use in the USA Several studies have beenconducted with these assays and the data indicate that theyhave similar performance characteristics as those marketed


elsewhere [28ndash30] and detect p24 approximately 5ndash7 daysafter the appearance of nucleic acid [3] Although these assaysare central lab-based assays they can be adopted to POCplatforms for use in resource-limited settings as they aremoresensitive and diagnose HIV infection earlier with a higherdegree of accuracy

232 Viral Load Technologies for Future Use The aboveNAT-based viral load systems described require testing tobe performed in a laboratory setting at a central or nationalreference laboratory by well-trained technicians Althoughthere are currently no POC viral load assays on the marketthere are a number of platformsassays in developmentDescribed below are a few viral load assays that are currentlybeing used and in the pipeline

24 NAT System (Alere Inc) The Alere NAT system is ageneric platform for the implementation of different NATassays with an integrated platform for quantitative measure-ment of HIV-1 and HIV-2 viral load from approximately25 120583L of whole blood The system detects HIV-1 Groups MN and O and HIV-2 The device on which the assay is run (aprototype of which is pictured below) has a small footprintis portable battery-operated and ruggedized to withstandharsh environments [3 31]

25 Liat Analyser (IQuum Inc) The Liat Analyser manu-factured by IQuum Inc is an automated sample-to-resultNAT platform that performs sample nucleic acid extractionpurification reverse transcription polymerase chain reaction(PCR) amplification and real-time detection to detect andorquantify pathogens Liat assays for HIV viral load testingand diagnostics have also been developed and independentlyevaluated by several laboratories To aid the operator andprovide reliable results the Liat Analyser incorporates a vari-ety of advanced features barcode data entry avoids errors insample or assay coding and on-screen prompts provide easy-to-follow directions to guide the operator through sampleloading and tube insertion [3]

26 EOSCAPE-HIV HIV Rapid RNA Assay System (Wave80 Biosciences) Based on its EOSCAPE technology Wave80 Biosciences is developing a rapid HIV NAT-based POCviral load assay designed for use in resource-limited settingsThe company describes the assay technology as incorporatingnon-PCR nucleic acid detection and quantitation fingerstickwhole blood processing within a single-use enclosed car-tridge Using a fingerstick lancet 100 120583L of whole blood isapplied directly into the cartridge and no external samplepreparation is required The sample is automatically pro-cessed in 45minutes the operator then inserts the processingunit into the reader for a quick 2-minute scan Equipped witha simple touchscreen interface the reader is capable of trans-mitting test results through wired and wireless connectivityThe EOSCAPE-HIV system will be capable of providingeither a qualitative or a quantitative HIV-1 RNA test result(detection threshold of 1000 copiesmL) in less than an hour[3]

27 SAMBA (Diagnostics for the Real World) The SimpleAmplification Based Assay (SAMBA) is being developed bythe Diagnostics Development Unit (DDU) at the Universityof Cambridge Two NAT-based HIV assays are being devel-oped (i) a semiquantitative test for monitoring of ART and(ii) a qualitative test for the use in EID The first SAMBAHIV assay to be launched will be the semiquantitative viralload assay The SAMBA machine will integrate extractionamplification and detection into a bench-top analyzer withamplification and detection taking place in a closed cartridge[3]

The SAMBA HIV test uses 200 120583L of plasma or 100 120583Lof whole blood Amplification is based on both target andsignal amplification The visual detection of the RNA orDNA target can be read off of a test strip visually within 25minutesThe test strip is based on a nitrocellulose membranein a dipstick format The SAMBA assay was able to detectall HIV-1 subtypes at 400 cpmL Currently however theSAMBA semiquantitative assay is calibrated to distinguishbetween patients with viral loads above or below 1000 cpmLThe total amplification time of the SAMBA is one hourwith throughput suitable for use at a small laboratorymdashfor example at district hospital level in sub-Saharan AfricaDiagnostics for the Real World Ltd the spinout company ofDDU located in California will be the manufacturer of theSAMBA system [3]

3 Additional Viral Load Technologiesin the Pipeline

The Cepheid GeneXpert System a fully automated andintegrated system for PCR-based nucleic acid testing isin use for detection of multiple pathogens and is beingdeveloped for HIV The Northwestern Global Health Foun-dation (NWGHF) in collaboration with Quidel Corporationis developing a POC rapid RT-PCR testing platform that willbe both easy to use and of low cost Lumora has introducedthe Bioluminescent Assay in Real-Time (BART) a platformfor performing molecular diagnostics that allows real-timeclosed-tube quantitative detection of amplification by usinga hardware system that can generate and store objectivetest results Advanced Liquid Logic Inc (ALL) providesdigital microfluidics technology solutions for liquid handlingapplications The company has developed a compact bench-top immunoassay analyzer (pictured right) that is currentlybeing evaluated The company indicates that it is evaluatingvarious assay formats as well as the portable analyzer Cavidiis developing a new automated platform for near-patient viralload monitoring The system will combine the strengths ofRT technology with the advantages of an automated walk-away platformThis should provide fast and robust viral loadmonitoring for all HIV types and subtypes [3]

31 Early Infant Diagnosis Diagnosing HIV infection earlyin infants can help in suitable therapeutic intervention whichgreatly reduces infant mortality due to HIV infection Cur-rent p24 immunoassays have improved sensitivity comparedwith previous p24 tests because of the inclusion of a heat


denaturation step for separating p24-antibody complexesthat commonly interfere with assay sensitivity Many studiessupport the use of the Perkin Ultra p24 immunoassay anddried blood spot analysis for early infant diagnosis (EID)However p24 immunoassays are not widely used owing toa lack of validation data Also because p24 immunoassaysrequire specialized equipment and skilled staff p24 rapidtests are preferred for EID in resource-limited locations[32] Development of p24 rapid tests has focused mainlyon improving sensitivity [33] In 2010 Parpia et al testeda microfluidic p24 test on 25120583L plasma samples from 389South African infants [34] Their design included a heat-shock component to break the antigen-antibody complexesThey achieved 95 sensitivity 99 specificity and a detec-tion limit of 42 500 RNA copiesmL The current prototypecan be performed within 20 minutes

32 Ultrasensitive p24 Antigen Assay (NWGHF) NWGHF isdeveloping an ultrasensitive p24 antigen rapid lateral flowassay for POCuseThe technology to be called Lynx involvesnot only a lateral flow strip that detects HIV p24 antigen butpreanalytical devices for separating plasma from heel-stickblood and disrupting immune complexes that would interferewith immunoassays NWGHF has demonstrated proof ofprinciple of the test The assay procedure involves collecting80 120583L of heel-stick blood from the infant using a capillarytube plasma is separated and the sample is subjected to ldquoheatshockrdquo in small battery-powered processor device the rapidtest strip is inserted into the device and after approximately30 minutes the result is read The total assay duration isapproximately 40 minutes In early testing the assay hasshown about 95 sensitivity and 99 specificity

33 PanNAT Diagnostic Platform (Micronics Inc) Micron-ics Inc a subsidiary of Sony Corporation has developed thePanNAT system which is a small portable microfluidic plat-form for POCuse in in vitromolecular diagnosis of infectiousdiseases in resource-limited settings It uses a fluorescent-based reader capable of processing individual disposableassay-specific cartridges each of which is designed to per-form a single- andor multiplexed nucleic acid assay Thereader includes all necessary reagents on board The systemis lightweight portable battery- or centrally powered WiFi-enabled capable of storing up to 350 test results beforeprompting the user to download or delete results and canprovide results within 30 to 40 minutes

Portable Tests Portable tests for HIV monitoring compriseCD4+ T-cell count assays and HIV quantification assaysCurrently only a handful of truly portable CD4+ T-cellcounters are available commercially PointCare NOW theCyFlow miniPOC (Partec) Pima Test (Alere) and DaktariCD4+ (Daktari Diagnostics) All are fully automated can bepowered by batteries or electricity use le 25 120583L of blood andprovide same-day results Additional POC CD4+ T-cell testsin development include a multiplex infectious disease test(MBio Diagnostics) and a semiquantitative electricity-freeCD4+ T-cell blood test (Zyomyx) [35] Both the PointCare

NOW and CyFlow miniPOC measure absolute CD4+ T-cellcounts and the percentage of T cells expressing CD4+ anddo not require cold chain for reagent storage The CyFlowminiPOC has a substantially high throughput with thecapability of processing 250 tests per day No independentdiagnostic data for either test were available at the time ofwritingThePimaTest andDaktari CD4+ are cartridge-basedtests in which a blood sample is inserted into a disposablecartridge containing the necessary reagents per testThePimaTest introduced in late 2010 has yielded results comparableto those of other ARTmonitoring devices [36ndash39] While themajority of Pima Test measurements underestimate CD4+T-cell counts one study demonstrated 963 sensitivity forconcentrations below a cut-off of 250 cells120583L [38] A recentstudy also found that Pima Test capillary blood samplesyielded less precise results than venous blood samples Thishighlights the need to address sources of error that candecrease a POC testrsquos accuracyNo portable PCR tests forHIVquantification are currently on the market but several are inthe pipeline

Several other POC tests that may be portable are alsounder development According to PATH a low-cost versionof a fully automated rapid PCR device is in developmentfor resource-limited settings [35] This test known as theLiat HIV Quant Assay (IQuum Inc Marlborough USA)uses whole blood in a single container for PCR amplificationand analysis in 88 minutes [40] The SAMBA HIV-1 testis a dipstick-based nucleic acid assay for HIV detectionand amplification After collection of viral RNA isothermalamplification of RNA is automated by amachineThe currentprototype tests one sample in lt2 hours but future prototypesare expected to test 5ndash10 samples simultaneously In onestudy SAMBA results agreed with RT- PCR results for 69clinical samples with different viral subtypes or viral loads(78 to 95 times 106 copiesmL) Additional advantages of thistest include (i) its lower cost because unlike PCR it requiresno thermo cyclers (ii) that it has multiplexing capabilitywhich permits detection of HIV subtypes and (iii) that itallows same-day diagnosis An additional SAMBA test forqualitative early infant diagnosis is also under developmentOther companies developing POC viral load tests includeAlere and Micronics Several other POC PCR platforms indevelopment might also be easily adapted for HIV-associatedhealth care [35]

4 HIV Screening in Resource-Limited Settings

The most ready-to-use types of rapid tests are lateral flowtests which do not require complex reagents In generalrapid tests are less expensive (less than 1 United States dollar[US$] per capita) portable and easy to use require few or noreagents or equipment and provide resultswithin 30minutesEach test rapidly detects antibodies against HIV from a smallvolume of plasma serum whole blood saliva or urine withhigh specificity and sensitivity The use of saliva and urinespecimens for rapid testing offers a discrete alternative toblood-based tests in settings where stigma lack of educa-tion cultural practices and privacy concerns undermine


HIV prevention [8] In addition the noninvasiveness ofspecimen collection eliminates the anxiety associated withblood collection and leads to higher rates of voluntary HIVtesting [41]

41 CD4 Counts The measurement of the absolute CD4 T-cell count is critical in the initial evaluation and staging ofHIV-infected persons HIV infects primarily immune CD4+T lymphocytes (CD4+ cells) [42] Antiretroviral therapyART especially when CD4+ cells are not yet depletedcan reduce viremia and slow its progression to AIDS [43]Furthermore if CD4 counts are established antiretroviralprophylaxis can reduce the risk of transmission in pregnantwomen to prevent mother-to-child transmission of HIV dur-ing pregnancy childbirth and breastfeeding [44]Therefore itis critical to performCD4+ cell counts upon diagnosis ofHIVand before initiating ART TheWHO currently recommendsART initiation if absolute CD4 counts are below 350cellsmm3 [44] The performance of the diagnostic platform withrespect to accuracy and precision is the most important forusing such technologies This is particularly challenging forCD4 testing platforms as ldquono gold standard technology orinternationally recognized reference preparation exists forCD4rdquo [45] Misclassification may delay the initiation of ARTor prophylactic treatment in some patients or treat largenumbers of patients who have CD4 counts above the ARTinitiation threshold

42 Existing CD4TechnologiesPlatforms There are currentlya handful of platforms that account for CD4 testing inresource-limited settingsThese are lab-based single platformsystems from BDBiosciences a division of Becton Dickinson(BD) BeckmanCoulter (Coulter)Millipore (formerlyGuavaand now a division of Merck) Partec and Apogee In thedeveloping world BD and Coulter are the most commonlyused platforms for CD4 testing [45]

43 Point of Care CD4 Testing Platforms The above-mentioned high- medium- and low-throughput platformsare systems primarily designed for use in laboratory settingsA number of them including the FACSCalibur Epics andFACSCount are used in developed as well as resource-limited settings The CD4 assays developed include selec-tive cell staining followed by capture or count by digitalphotography measuring CD4 molecules instead of cells ormeasuring proxy molecules of CD4 Some of the POC tech-nologies are already in use They include PointCare NOWthe Pima CD4 Analyser and the CyFlow CD4 miniPOCThe remaining technologies including Daktari BurnetZyomyxMBio and others are not yet commercially available[3]

44 Other Possible CD4 POC Tests In addition to thePOC CD4 testsdevices discussed above there are a fewother research and development groups working on plat-formsdevices that could potentially be used for CD4 count-ing

45 Automated POC CD4 Test Devices There are severalcommercial POC CD4 counting devices that are now avail-able and some are under development Most of them usefinger-stick blood samples which are rapid are robust haveflexible power options and utilize stable dried reagentsenclosed in a single test cassette or device The devices aredesigned for minimal operator use with varying through-put result outputs The PointCare NOW and the CyFlowminiPOCaremodified flow cytometersThePointCareNOWis the only POC CD4 device commercially available Thesystem is significantly heavier than the other technologiesmaking it less portable and venipuncture is required for largerspecimen volumes which may limit its use in low resourcesettings where phlebotomists are scarce However additionalequipment such as pipettors is not needed for operation andtherefore supply of other critical reagents is not a factor

A CD4 POC test marketed by Alere (Waltham MA) thePima CD4 test is the first commercially available automatedCD4 counting method that does not use traditional flowcytometric approaches The Pima utilizes dual-fluorescenceimage analysis to count CD3+ and CD4+ using labeledanti-hCD3 and anti-hCD4 The Pima cartridge contains allreagents and specimen during processing The Pima readersupplies the pumping functions and images the results todetermine colocalization of CD3+ and CD4+ cellsThe resultexpressed as the absolute number ofCD4+T lymphocytes per120583L and the CD3+CD4+ ratio is displayed by the instrumentalong with quality control results Cartridges using beadsthat represent ldquoNormalrdquo and ldquoLowrdquo counts are intended fordaily quality control testing of the PIMA instrument Recentevaluations in Zimbabwe andMozambique have shown goodperformance in comparison to flow cytometry and userassessment of the test protocols that suggest that the Pima hasthe potential for use at the POC [36 46]

Daktari Diagnostics Inc (Boston MA) is develop-ing a CD4 test that uses a novel microfluidic affinity-chromatographyshear-gradient technique to differentiallycapture CD4+ cells from whole blood and a unique nonop-tical detection to count them The test consists of a readerand a disposable cassette which contains all of the reagentsstabilized in blister packs Here flowing CD4+ cells adhereto an antibody-coated chamber while larger-sizedmonocytesare subject to large shear forces and cannot bindThe capturedCD4 cells are then lysed and the release of cellular ions ismeasured by impedance spectroscopy [47] The decrease inimpedance in the chamber due to CD4+ cell binding hasbeen shown to correlate with cell count over three orders ofmagnitude including 350CD4+mm3 the threshold forART[48]

mBio Diagnostics Inc (Boulder CO) is developing aCD4+ cell-counting system SnapCount It is a static two-color fluorescence imaging cytometry system composed ofsingle-use disposable cartridges and a simple reader withon-cartridge immune staining of whole blood samples Theinstrument addresses the high cost of conventional opticalsystems by using LightDeck technology a fluorescence assayillumination 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


electronics As the instrument is only utilized in the read-ing step multiple cartridges can be processed in parallelproviding a throughput of eight to ten samples per hourThis relatively high sample throughput might allow remotehealth care settings to meet their greater demand with fewerinstruments [49]

46 Nonautomated CD4 Test Devices In 2005 the CD4initiative held by Imperial College London (funded by theBill amp Melinda Gates Foundation) partnered with industrialand academic teams to develop power-free POC CD4 tests[50] and three devices are now being further developedfrom this project The Burnet Institute CD4 test device is asemiquantitative immune chromatographic strip (ICS) SomeICS assays have been shown to be effective tools for POCdiagnostics in RLS they are small of low cost have acceptableperformance and reagent stability and have a test formatthat is familiar to the intended user group The Burnet CD4test has been incorporated into a lateral flow strip (similarto an HIV rapid diagnostic test) with traditional rapid testformat including monocyte removal pad and immunogoldIn this device the transmembrane domain of cellular CD4is recognized by biotin-labeled anti-hCD4 which is detectedby a colloidal gold labeled anti-biotin A capture stripe ofanti-CD4 is adjacent to a reference stripe of a biotinylatedsurrogate protein The CD4 count is determined by the userto be either greater or lower than the reference stripe

Beckman Coulter has been developing a similar semi-quantitative ICS format to enumerate CD4 at the POCZyomyx Inc (Hayward CA) has developed a novel beadsedimentation system to count CD4+ cells [19] The reagentsinclude high-density anti-hCD4 particles as well as anti-hCD14 magnetic beads The device uses sedimentation ofhigh-density bead-associated CD4+ cells as a measurementprinciple The height of the sedimented and visible beadcellcolumn in a capillary is directly proportional to the number ofCD4+ cells when viewed against a precalibrated scale muchlike reading a thermometer [19]

There is no technology currently in widespread use forcountingCD4 at the POCMost of the technologies describedabove have incorporated the basic tenets for POC in termsof instrument size specimen volume reagent stability inde-pendent power supply and simple user interface Howeverother factors such as throughput associated consumables andreagents user training robustness of design and the methodof analysis vary considerably

Three assays using either scaled flow cytometric methods(PointCareNOW and the CyFlow miniPOC) or novel tech-nologies (the Alere Pima) are also in use Flow-based systemshave the advantage of established technology and have beenthoroughly evaluated in larger scale formatsThus evaluationof the new instruments will focus on improving and demon-strating the robustness and the ease of use in RLS The needfor appropriate quality controls (QC) and POC testing is anarea where QC is essential to ensure that specimen prepara-tion equipment and reagents are functional for user com-pliance with the protocol The Pima incorporates QC indi-cators for specimen collection and reagent and equipment

function The devices being developed by Daktari and mBiooffer cassette-based instrumented readers for increased accu-racy with a simple user interface and single or no movingparts This simplified instrumentation is anticipated to costless than other CD4 instruments in addition to havingpotentially lower failure rates and reliance on maintenance[49]

47 Viral Load Testing Technologies Overview Viral loadtesting is the method favored for monitoring HIV patientsonce they have been initiated onto ART High levels ofHIV circulating in the bloodstream indicate that the virusis actively replicating and these levels can be used with theaid of molecular methods to provide important informationregarding the risk of disease progression and to predict theoutcome of infection [19] Viral load testing is complicatedby HIV diversity and certain practical challenges includinglaboratory infrastructure and transport of samples

HIV is classified into two HIV-1 and HIV-2 major typesOf the two types of HIV HIV-1 is predominant and has beenmost responsible for the HIV pandemic that exists today [19]HIV-1 is divided into four groups designated byM N O andP the main group of which is group M Within the HIV-1groupM nine clades are recognized designated by the lettersAndashD FndashH J and K Recombinants between different HIV-1groupM subtypes are designated as either circulating recom-binant forms (CRFs) if fully sequenced and found in threeor more epidemiologically unlinked individuals or as uniquerecombinant forms (URFs) if not meeting these criteria [51]Up to 55 CRFs have been described so far [51 52] SomeCRFshave recombined further with other subtypes or CRFs givingrise to the so-called second-generation recombinants (SGRs)

The enormous diversity found inHIV as discussed earlierand extraordinary ability of this virus to evolve continuouslypose great diagnostic challenges The high level of geneticheterogeneity of HIV-1 and the emergence of recombinantstrains of the virus complicate viral load assay developmentIn an ideal world viral load assays would detect and quantifyall known HIV-1 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-1 subtypes Therefore it is important to consider theprevalence of HIV-1 and HIV-2 groups and subtypes in aparticular geographical region when choosing a viral loadassay

48 PCR-Based Diagnostics In response to the HIVAIDScrisis access to antiretroviral therapy (ART) has increaseddramatically over the past decade in low- andmiddle-incomecountries [53] However successful management of HIVrequires that patients receiving ART be monitored routinelyto assess treatment efficacy and detect treatment failure dueto drug resistance and other causes Unfortunately currentlaboratory-based methods to monitor ART are unaffordableunavailable or inappropriate for low-resource settings [54]

Rapid antibody tests are widely available in developingnations but they cannot be used to monitor HIV progressionor treatment efficacy The standard of care to monitor ART


is quantitative viral load testing based on plasma HIV RNAconcentration [55] Although CD4 count has also been usedto monitor ART recent studies suggest that it may not detectearly treatment failure adequately [56] The gold standardmethod for viral load testing RT-qPCR is unsuitable forsettings where trained technicians expensive reagents elec-trically powered equipment and dedicated laboratory spaceare often unavailable Therefore a viral load test that isappropriate for such settings is needed

The NATs have the potential to offer many advantages atthe POC such as low limits of detection and quantificationof the level of infection PCR is an enzyme-driven processfor amplifying short regions of DNA in vitro It can createmillions of DNA copies by cycling between different tem-peratures to allow repeating steps (denaturation annealingand elongation) of DNA replication to take place Despitethe simplicity and amplification power of PCR chemistrylimitations in its supporting hardware still hinder PCR fromreaching its full potential In particular improvements inthermal cycling speed instrument size and reaction volumeare still much needed The 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 for HIV infec-tion at POC and this has significantly expanded diagnosticcapabilities of testing sites in developed countries as wellas resource-limited settings Despite advances made by thewidespread availability of rapid tests all antibody-baseddetection of HIV exhibits some limitations HIV-specificantibody typically begins to appear around three weeks afterinfection allowing for detection by most antibody-basedassays within 3ndash6 weeks [57] This window period prior toor during early sero conversion may lead to false-negativetest results in recently infected individuals Additionallyaccurate diagnosis of infants born to HIV-infected motherscan be challenging if based solely on antibody positivity sincevertically transferred maternal antibodies may persist for 12ndash18months after birth [58] For confirmatory diagnosis of earlyHIV infection or infant diagnosis nucleic acid amplificationtests (NAAT) are preferred as HIV-1 RNA can be detectedas early as 10ndash12 days after infection and HIV-1 DNA andorRNA are definitive indicators of active infection [59] Intheir current form however NAATs are not feasible for POCtesting because they are time consuming expensive andtechnically complicated

5 Current Isothermal AmplificationTechnologies

51 Isothermal Amplification Isothermal amplification tech-niques operate at a single temperature eliminating theneed for a thermocycler enabling them to be conductedon simple and portable heating systems Several isothermalamplification techniques have been developed in recentyears such as helicase-dependent amplification rolling circleamplification and nicking enzyme amplification reaction[60ndash62] Loop-mediated isothermal amplification (LAMP)

[10] has been optimized for the detection of DNA andorRNA (RT-LAMP) from a wide range of bacterial and viralpathogens including HIV [63ndash65]

LAMP or RT-LAMP exhibits several characteristics thatare ideal for integration into a rapid nucleic acid-based diag-nostic test The amplification reaction requires six primersspecific for eight separate regions within the target sequencecontributing to the high specificity of the amplificationmethod Amplified material can typically be detected within15ndash60 minutes when incubated at a constant reaction tem-perature of 60ndash65∘C [66] LAMP has also proven to beless sensitive to biological inhibitors than PCR [67] whichenables direct amplification from clinical specimens therebyeliminating the need for an additional nucleic acid extractionstep Direct amplification from plasma whole blood and oralfluid has previously been demonstrated forHIV-1 [63 64 68]Lastly immediate visual detection of amplified products isfacilitated by the large amount of DNA that is generated byeach reaction Several groups have incorporated fluorescentdetection methods into the LAMP assay for real-time orimmediate naked-eye detection [64 66 69]

52 Nucleic Acid Sequence-Based Amplification (NASBA)The NASBA techniques like transcription mediated amplifi-cation (TMA) and self-sustained sequence replication (3SR)mimic in vivo retroviral replication mechanisms to produceRNA amplicons from an RNA template Modified cDNAis formed from an RNA template which is then rapidlyamplified into RNA amplicons The complete process is asingle step and proceeds in a single volume with an ssRNAproduct suited for direct use with hybridization probes mak-ing NASBA very appealing for point-of-care Technologies(POCT) [70]

The target RNA being labile calls for faster and carefulsampling 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-specific amplification making robust primer design andassay evaluation crucial [71] Furthermore an initial 95∘Cstrand separation step is required if dsDNA is to be targetedwhilst RNA amplification requires a 65∘C step to removesecondary structures These temperature steps will be anengineering consideration in POCT devices where precisethermal control and associated increased power consumptionwill be disadvantageous

53 Helicase-Dependent Amplification (HDA) Thehigh tem-perature requirement to separate dsDNA can be overcome byusing stand separating enzyme helicase in PCR amplificationof DNA The typical existing HDA protocol needs 60ndash120min for low copy number targets This shortcoming canbe overcomeby including the use of restriction endonucleasestargeting regions upstream of the target sequence to enhancehelicase activity in the target region addition of crowdingagents and increasing enzyme concentrations [20]TheHDAis appealing for POCT as it requires single set of primersand two enzymes (three for reverse transcription-HDA) and


is compatible for existing fluorescent detection chemistries[20]

531 Recombinase Polymerase Amplification (RPA) RPA isa single tube single temperature (37ndash42∘C) amplificationmethod The key to the amplification process is the for-mation of a recombinase filament a complex combining atarget-specific primer and a recombinase enzyme When thetarget-specific sequence is encountered by the recombinasefilament it performs strand exchange inserting the primeronto the target The displaced d-loop formed is stabilized byssDNAbinding proteins (gp32) to prevent reannealing Spon-taneous disassembly of the recombinase filament upon strandexchange leaves the primertarget hybrid open to extensionby strand-displacing polymerase activity Repetition of thecycle leads to geometric amplification Favorable thermalrequirements procedural simplicity and very rapid ampli-fication (20ndash40min) make this recently developed process[72] a leading technology for integration into POCT devicesThe added ldquooff-temperaturerdquo ability of RPA to proceed ata variety of temperatures is of great appeal for field applica-tions where precise temperature control is often technicallychallenging and will allow for instrument-free amplificationThe biochemistry of RPA is incompatible with existingintercalating dyes molecular beacons and TaqMan technol-ogy Alternative fluorescent probe detection strategies likeTwist-Ampexo and TwistAmp have been developed to allowsingle tube fluorescent detection using sequence-specificprobes As an emerging technology there is comparativelylittle in the published literature regarding RPA technologyRPA primerprobe design and its integration with POCTdevices

532 Loop-Mediated Isothermal Amplification (LAMP)Among the isothermal nucleic acid methods currentlyavailable loop-mediated isothermal amplification [73](LAMP) is the most widely researched and has beenwell characterized offering significant support during thedevelopment process LAMP is a rapid amplification methodemploying a strand displacing Bst DNA polymerase and4ndash6 primers two of which are ldquofold backrdquo primers [74]which form stem-loops motifs with self-priming capabilityThis results in an amplification scheme where the primingsequence is copied with each round of replication andremains tethered to the previous amplicon resulting in aconcatenated product of alternating senseantisense repeatsof varied length Subsequent studies have found that theuse of additional ldquoloop primersrdquo which bind to the loopstructures can greatly reduce the reaction times resultingin a total of 6 primers [75]

The 60ndash65∘C reaction temperature combined with aminimum of 4 primers makes LAMP a highly specificreaction allowing an ldquoamplification is detectionrdquo schemeThisspecificity has allowed the insoluble pyrophosphate reactionby-product [76] to be employed in a turbidimetric detectionstrategy for both qualitative visual indication [77] or real-timequantitative turbidimetry [78] which offers a very simplerobust detection strategy for POCT integrationWhilst a 95∘C

initial strand separation step is not essential [79] it has beenshown to increase analytical sensitivity [80] As with someother isothermal methods (SMAP NEAR and SDA) LAMPis highly dependent on the careful design ofmultiple complexprimers and this can be overcome by the use of appropriatesoftware [74]

533 Rolling Circle Amplification Technology (RCA) RCA isa powerful technique which exploits the strand displacementand highly processive polymerase activity of the Phi29 bac-teriophage DNA polymerase (Q29DNAP) acting on circularDNA targets [81 82] The basic RCA reaction (linear RCAor single primer RCA) is initiated by a primer annealingto a circular ssDNA The Q29DNAP can elongate a newstrand of the circular template eventually completing a loopand reaching the point of initiation Strand displacementactivity allows the newly forming strand to continuouslydisplace the previously generated strand as polymerizationadvances Generation of a continuous catenated ssDNA ofup to 05 mega bases [83] has been reported and continuesuntil an external factor such as nucleotide depletion haltsthe reaction This continuous catenated product attached tothe template allows in situ or localized amplification whichcan be used to concentrate labels within a small detection areaand enumerating single DNA molecules [61 84 85]

534 Single Primer Isothermal Amplification (SPIA) SPIA isa linear amplification technology for DNA based on repeatedreplication of target sequences enabled by the use of chimericRNADNA primers which bind target regions and initiatepolymerization [86] The RNADNA primer is engineered insuch a way that RNase H degradation of the RNA portion ofthe chimeric primer will reexpose the binding site to allow asubsequent primer to anneal Strand displacement activity ofthe polymerase removes the previously generated strandThisrepeated cycle continuously generates new amplicons untilreagents or primers are depleted A similar method Ribo-SPIA developed to amplify total mRNA replicates only theoriginal transcripts and not copies resulting in a high-fidelityproduct SPIA will become more widespread as genomicanalysis enters the point-of-care domain

535 Smart Amplification Process Version 2 (SMAP2Smart-Amp2) This nascent amplification technology (not to beconfused with the signal amplification method SMARTsignal-mediated amplification of RNA technology) employssimilar enzymes and self-priming loop motifs to LAMPIn contrast to the symmetrical primers of LAMP SMAP2primers are designed asymmetrically with different tailmotifs in the two target flanking primers This serves toreduce the formation of background products from misam-plification The amplification process occurs in two stepsan initial ldquokey intermediaterdquo step forming a target sequenceflanked 31015840 and 51015840 with fold-back domains to provide self-priming ability and a second amplification step where the keyintermediates undergo repeated self-priming and rapid targetamplification resulting in concatenated primer inclusiveamplification products


In addition to this amplification format SMAP2 employsbackground suppression technology to increase specificityand permit an ldquoamplification is detectionrdquo assay Ultrahighspecificity is achieved by employingThermus aquaticusMutSto identify mismatched primertarget hybrids MutS scansdsDNA and will irreversibly bind to any mismatch duplexwith single nucleotide sensitivity [87] Bound MutS preventspolymerization thus checking amplification of nonspecificsequences resulting in complete inhibition of nonspecificamplification The incorporation of MutS and asymmet-ric primer design permits single nucleotide discriminationmaking SMAP2 particularly useful for SNP identificationWhilst SMAP2 has been shown to proceed slower thanLAMP the SMAP2 techniquersquos very high specificity highsensitivity (3 copies) and powerful amplification (100-foldlarger than PCR) combinedwith the developers (DNAFORMandRIKEN Yokohama Japan) reporting of specific detectionfromcrude cell lysatemake this a promising development andpotentially powerful tool for POCT devices [88 89]

536 Strand Displacement Amplification (SDA) Thismethod relies on bifunctional primers incorporating bothtarget recognition and endonucleases regions [90 91]Following strand separation these bifunctional primersextend incorporating the restriction target into the ampliconBumper primers which bind and extend upstream releasethis amplicon Successive rounds of primer binding generatedsDNA incorporating restriction sites which can then beacted upon by the restriction endonucleases to nick a singlestrand of the newly formed duplex This nicking allows thepolymerase to displace the existing strand and incorporatea new amplicon This nick and run scheme is repeatedto effect exponential amplification Single strand nickingis effected by the incorporation of a modified adeninenucleotide dATP aS (59-O-l-thiotriphosphate) which isresistant to the endonuclease activity Thus only the newlyincorporated primer will be nicked leaving the amplifiedstrand to repeatedly act as a template for primer bindingThecomplex asynchronous reactions occur concurrently anduser interventions are limited to an initial heat denaturationwith primers followed by addition of polymerase andrestriction enzymes at a 37 uC190 incubation a protocolwhich is by no means complex and appears amenable toPOCT use [91]

537 Nicking and Extension Amplification Reaction (NEAR)NEAR is a recent development of the earlier describedEXPAR reaction [62] Capitalizing on nicking enzymes toexpose binding sites for primers the EXPAR displays excel-lent reaction kinetics and 106- to 109-fold amplification ina few minutes However EXPAR is limited to amplificationof sequences adjacent to native nicking enzyme recognitionsites within the target genome [92] NEAR is a refinementof EXPAR to allow amplification of any target by insertingnicking-enzyme recognition sites adjacent to target regionsThe two-stage NEAR reaction proceeds in a similar mannerto the SDA reaction exploiting nicking-enzymes to generatea site from which polymerase elongation can initiate In

contrast to SDA the nicking enzyme employed in NEAR willonly nick a single side of a duplex removing the need asseen in SDA for strand modification of the duplex to preventdouble stranded cleavage

538 Isothermal and Chimeric Primer-Initiated Amplificationof Nucleic Acids (ICAN) ICAN is a simple scheme forDNA amplification at 55∘C using relatively few reagentsa pair of 51015840-DNA-RNA-31015840 chimeric primers thermostableRNase H and a strand-displacing DNA polymerase [93]Following initial heat denaturation of the target dsDNAthe chimeric primer binds to the template and is elongatedby BcaBEST DNA polymerase The newly formed strandis nicked by thermostable RNase H action not at the 31015840border of the chimeric primer as initially thought but at thepenultimate 31015840 RNA residue allowing the strand displacingDNA polymerase to release a newly synthesized strand witha single 51015840 RNA residue leaving the template with a truncatedprimer which is still sufficient to prime elongationThe cyclerepeats until the chimeric primer is sufficiently shortenedallowing a new free chimeric primer to anneal preferentiallyrecommencing the cycle [94]

In addition to this multipriming model a templateswitching mode of amplification has been identified [95]Template-switching amplification occurs when both forwardand reverse primer bind to the same dsDNA target andproceed to elongate toward one another eventually switchingthe template from using the original template to usingthe newly synthesized strand elongating from the oppositeprimer as the template This displaces both parent strandsforming a dsDNA of two daughter strands consisting ofthe target flanked by the primer regions with incorporatedchimeric primer on one strand This dsDNA of daughterstrands is acted upon by RNase H which introduces a nick inthe RNA region and polymerase elongation can commenceIf both forward and 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 Ifthe nicking and elongation occur asynchronously a single-stranded product is formed still having an incorporatedchimeric primerThis single strand with primer can enter themultipriming amplification cycle thus there is no dead-endproduct and amplification will be sustained until the reagentsbecome exhausted

539 Conclusion Many of the isothermal amplificationmethods described above display speed amplification powerand analytical and diagnostic specificity and sensitivity equalto and often in excess of existingmolecular techniques basedon real-time PCRqPCR Given this excellent performanceand their suitability for miniaturization it is highly likelythat isothermal amplification strategies will become com-monplace in the next generation of point-of-care diagnosticdevices Although tremendous progress has been made sincethe discovery of PCR still there is no robust nucleic acid-based point of care PCR


6 Microfluidic Diagnostics

This section categorizes various fluidics technologies suchas pressure-driven flows capillary flows electromagneti-cally driven flows centrifugal fluidics acoustically drivenflows and droplet fluidics Then three broad categories ofPOC microfluidic testing devices are considered lateral flowdevices desktop and handheld POC diagnostic platformsand emergent molecular diagnostic POC systems Suchevolving trends as miniaturization multiplexing network-ing new more sensitive detection schemes and the impor-tance of sample processing are discussed It is concluded thatPOC microfluidic diagnostics have a potential to improvepatient treatment outcome and bring substantial savings inoverall healthcare costs

Microfluidic diagnostics had an explosive growth in thelast 20 years spurred by the convergence of clinical diagnostictechniques (such as blood gas analysis immunoassays andmolecular biology testing) and mature micro fabricationtechnology that allowed production of sub-millimeter-sizefluidic channels and reservoirs in a variety of material sys-tems (eg silicon polydimethylsiloxane (PDMS) and poly(methyl methacrylate) (PMMA)) [96]

61 Microfluidic Technologies Microfluidic diagnostics usetechnologies to accomplish a predetermined set of operations(ie to bring the sample and reagents together to add bufferto implement wash and to facilitate the readout) requiredby the specific biochemistry of the tests and detection tech-niques They can be classified according to fluid propulsionsuch as pressure-driven flow and electromagnetically drivenflow They can also be classified according to the type offlow like continuous flow or the so-called segmented flow(where fluid is advanced in discrete packets or droplets)The segmented flows (also called droplet microfluidics) anextremely important emerging technology can be achievedon a variety of platforms for example centrifugal platformspressure driven platforms and electromagnetically drivenflows

Strip-based tests that use capillary forces are among themost ubiquitous and commercially successful POC testsThere are many evolving LF testing technologies like nucleicacid hybridization-based LF devices [97] or a combination ofantibody-antigen recognition with nucleic acid hybridizationin nucleic acid lateral flow immunoassays [98] and utilizationof advanced labels such as resonance-enhanced absorp-tion [99] chemiluminescence [100] upconverting phosphors[101] silver-enhanced gold nanoparticle labels [102] Othertrends involve developing a larger number of immunoassays[103] advancing quantification of the detection [104] andquality control in manufacturing to increase reproducibilityof tests

62 A Lateral Flow Assay for Quantitative Detection ofAmplified HIV-1 RNA Rebecca R Richards-Kortumrsquos groupfrom Rice University Houston TX have described a lat-eral flow assay that employs gold nanoparticle probes andgold enhancement solution to detect amplified HIV RNA

quantitatively They showed coupled with nucleic acidsequence-based amplification (NASBA) that this assay candetect concentrations of HIV RNA that match the clinicallyrelevant range of viral loads found in HIV patients Theirlateral flow assay achieved a resolution of 05 log

10copiesmL

over a linear range that extends 25 orders of magnituderanging from 105 to 13 log

10RNA copiesWhen coupled with

NASBA the LFA can detect 50 copies of HIV gag RNA Wespeculate that this LOD corresponds to a plasma viral loadof roughly 1000 copiesmL assuming that a plasma samplevolume of 100mLwill be used and that half of viral RNA fromthe sample is added to the NASBA reactionThe performanceof the LFAwhen detecting NASBA products suggests that theLFA may be sufficient to detect significant changes in viralload suppression of viral replication and therapeutic failureThe LFA only requires a heat block scanner or camera andpipetteThe lateral flow assay uses a sample volume of 20mLrequires only three steps over the course of 20 minutes costs$080 per strip using commercial reagents and performsconsistently after short-term storage By modifying the targetcapture positive control and probe sequences the LFA maybe adapted to detect other RNA targets The LFA is capableof detecting short amplified sequences or long genomicsequences The LFA is suitable for low-resource settings andhas the potential to be performed at the POC [105]

63 Miniaturization of NAT Instrumentation Miniaturiza-tion of NAT instrumentation has been the focus in recenttimes Genie II (OptiGene Horsham UK and Twista(TwistDx Cambridge UK) demonstrates the possibility formore portable NAT equipment [106] Separate amplificationand detection of the PCR product allow for greater sensitivityand elimination of nonspecific signals With regard to POCTapplications the requirement of multiple procedural stepsfor amplification and separate detection is cumbersomeHowever development of simplified assays is limited by theneed to maintain analytical sensitivity and specificity in aprotocol consisting of fewer steps

64 Combined Amplification and Detection The advent offluorescent DNA probes and intercalating dyes [107 108] hasallowed the real-time quantification of amplification productsin both PCR and isothermal amplification reactions Thelimiting factor of such systems is the need to maintainanalytical sensitivity to the target whilst developing reactionconditionswith sufficiently high analytical specificity to elim-inate nonspecific products accumulating and producing afalse-positive result The use of sequence-specific fluorescentprobes such as TaqMan and molecular beacons offers thebenefits of combined amplification and detection in reactionslacking the specificity for nonspecific detection methodsProbe-based real-time detections also allow for multiplexingusing different probe fluorophores The simplicity of com-bined amplification and detection in a single step is desirableand will hasten the time to result

65 Amplification-Free Direct Nucleic Acid Detection Suchtechniques use highly sensitive detection strategies to identify


target sequences in a sample without the need for nucleic acidamplification This offers the potential for simplified proto-cols reduced reagent consumption and simplified operatingplatforms The success of such systems is entirely dependenton the development of robust biosensors with analyticalsensitivity sufficient to detect the very low abundance NAin complex clinical samples whilst maintaining appropriateclinical specificity for diagnostic use Non-POCT systemsincorporating direct NA detection are commercially avail-able For example NanoString (Seattle USA) offers a bench-top instrument using molecular barcodes for gene analysisPortable systems for direct detection such as those beingdeveloped by Genefluidics (Irwindale USA) using electro-chemical detection and ExoCyte (Reading UK) employingsilicon nanowire carbon nanotube and quantum dot tech-nologies have yet to be cleared for diagnostic use Directdetection technologies are expected to enter the global testingmarket within the next 10 years and have the potential todisplace numerous methods currently in use [106]

7 Paper-Based Sensors

Paper is an extremely versatile material possessing desirableproperties such as mechanical flexibility and strength Themain constituent of paper is cellulose fiber and this can behighly attractive for certain applications as it allows liquidto penetrate within its hydrophilic fiber matrix without theneed of an active pump or external source [109] More-over cellulose fibers can be functionalized thus changingproperties such as hydrophilicity if desired as well as itspermeability and reactivity It can be produced sustainablyand inexpensively is ubiquitous globally and provides anexcellent substrate for chemical fictionalization making itwell suited to use in low-cost point-of-care (POC) diagnosticdevices In recent times paper has been employed in theconstruction of more complicated analytical devices knownas lab-on-a-chip (LOC) Conventional LOC devices oftenmade of glass or PDMS while considerably more simplifiedthan their full-scale laboratory counterparts still maintain acost which is prohibitive to their deployment in economicallydeveloping regions due to the specialist equipment and cleanrooms required for their manufacture [110] In 1956 the firstpaper device for the semiquantitative detection of glucosein urine was demonstrated [111] that further developed intoimmune chromatographic paper test strips (also known aslateral flow or dipstick tests) with the pregnancy test kit beinga well-known example [103] Paper has been suitably modi-fied to detect analytes by different methods like colorimetricelectrochemical electrical conductivity chemiluminescenceand electro-chemiluminescence [112]

Paper-based sensors are a new alternative technology forfabricating simple low-cost portable and disposable analyti-cal devices for many application areas including clinical diag-nosis food quality control and environmental monitoringThe unique properties of paper which allow passive liquidtransport and compatibility with chemicalsbiochemicals arethe main advantages of using paper as a sensing platformDepending on the main goal to be achieved in paper-based

sensors the fabrication methods and the analysis techniquescan be tuned to fulfill the needs of the end user Currentpaper-based sensors are focused on microfluidic deliveryof solution to the detection site whereas more advanceddesigns involve complex 3D geometries based on the samemicrofluidic principles Although paper-based sensors arevery promising they still suffer from certain limitations suchas accuracy and sensitivity However it is anticipated that inthe future with advances in fabrication and analytical tech-niques there will be more new and innovative developmentsin paper-based sensors These sensors could better meet thecurrent objectives of a viable low-cost and portable devicein addition to offering high sensitivity and selectivity andmultiple analyte discrimination

71 CMOS Cell Sensors for Point-of-Care Diagnostics Com-plementary metal-oxide-semiconductors- (CMOS) basedproducts can enable clinical tests in a fast simple safeand reliable manner with improved sensitivities Extensiveuse of CMOS-based sensors in DNA diagnostics [113 114]and commercialization of CMOS-based DNA sequencingsystems (Ion Torrent Systems) [115] is already promising forthe involvement of CMOS-based cell detection systems inmedical applications

72 Challenges in Paper Platforms Although there is enor-mous potential in paper as a platform for LOC devices thereis a lot of scope to improve in terms of sensitivity This istypically due to how the sample is introduced onto the paperwhich can be at a considerable distance of 10 to 15 millimetersfrom the detection zone As the analyte is delivered across thepaper the local analyte concentrationmay decrease as a resultof solution spreading and may also evaporate if the distanceand time of travel are far from the point of introduction Theother problems are encountered inmultiplex analysis as thereis the potential for cross-talk as some of the signal reportersmay diffuse to neighboring channels [112] There are limitedchemistries available for the conjugation of biomolecules tocellulose for use in biomedical applications

73 HIV Subtypes Isolation on Chip Utkan Demirci grouphave demonstrated a microfluidic chip device that can effec-tively capture various subtypes of HIV particles throughanti-gp120 antibodies which were immobilized on themicrochannel surface They showed that Protein G-basedsurface chemistry has a better control over the antibodyorientation on the surface compared to antibody immo-bilization methods including passive adsorption covalentbinding and NeutrAvidin-based surface chemistry Thisplatform technology can be potentially used tomeasureHIV-1 viral load in resource-constrained settings Protein G-basedsurface chemistry when used together with NeutrAvidin-based surface chemistry enables the separation of captureand detection chemistries that can potentially reduce thenonspecific binding and enhance detection outcomes Theirimmunosensing device enables the development of POC on-chip technologies to monitor viral load and guide antiretro-viral treatment (ART) in resource-constrained settings [116]


8 Fraunhofer ivD-Platform

A consortium of seven Fraunhofer Institutes have developeda lab-on-chip system called ldquoFraunhofer ivD-platformrdquowhichcan be potentially used as POC diagnosticsThe platform fea-tures a high degree ofmodularity and integrationModularityallows the adaption of common and established assay typesof various formats Integration lets the system move fromthe laboratory to the point-of-care settings with multiplexingcapability By making use of the microarray format the lab-on-chip system also addresses new trends in biomedicineThe low-cost device has reagents reservoirs microfluidicactuators and various sensors integratedwithin the cartridgeIn combination with fully automated instrumentation (read-out and processing unit) a diagnostic assay can be performedin about 15 minutes This is possible with user-friendly inter-facing read-out unit data acquisition and data analysis unitstogether

The assays for nucleic acids (detection of 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 internalreflectance fluorescence (TIRF) Furthermore integration ofsample preparation and polymerase chain reaction (PCR) on-chip has been done The instrument is capable of providingheating-and-cooling cycles necessary for DNA amplification[117]

9 Detection

Electrochemical and optical technologies are the clear leadersin detection technologies in the market as they do not usecomplicated instrumentation for detection Therefore point-of-care detection should be able to detect signal withoutusing any complicated instrumentation or if the results canbe visualized

91 Optical Detection Optical detection is the simplest andmost popular method used in immunoassay applicationsMost immunosensors are based on optical detection andcommonly use a label (eg a fluorescent label enzyme ormetallic particle) that may facilitate optical signal enhance-ment and increase detection sensitivity Optical immunosen-sors combined with microfluidic chips have recently beenproposed as an attractive immunosensing platform andmany reviews have explored their potential applications inclinical diagnostics particularly because there is a growingneed to simultaneously screen multiple proteins in a singlesample The optical detection method which can be easilyimplemented in microfluidic systems is a prime candidatefor this multiplexed analysis Optical detection methods canbe divided into five main categories on the basis of detec-tion signal fluorescence luminescence absorbance (col-orimetry) surface plasmon resonance (SPR) and surface-enhanced Raman scattering (SERS) each technique hasinherent advantages and disadvantages In this section wefocus on the recent multiplexing applications in microfluidicimmunosensors using optical detection techniques

There are several optical detectionmethods used for POCapplications such as fluorescencewith variants such as Forsterresonance energy transfer (FRET) and upconverting phos-phor technology luminescence absorbance (colorimetry)surface-plasmon resonance (SPR) and various categoriesof light scattering Rayleigh (particles much smaller thanwavelength) Mie (particles comparable to wavelength shapedependent) geometric (particles larger than wavelength)resonant (wavelength overlaps an electronic transition ofthe particle) and Raman (vibrational quanta added to orsubtracted from the excitation wavelength) [118] The mostcommonly used technique is absorbance as it is commonlyused in LFAs based on gold or polymer (nano-) particlesHowever fluorescence is used for the broadest range ofdifferent types of POC assays [119] for reasons of sensitivityand more recently the ready availability of a range ofdifferent colors of efficient fluorophores including quantumdots quantum-dot barcodes and fluorescent nanoparticlesproviding improved limits of detection Fluorescence in somecases can detect single particle LODs enabling multitargetmultiplexing [120]

The supercritical angle fluorescence (SAF) which is beingused recently detects fluorescence emitted in close proxim-ity to a fluorophore-supporting optically transparent chipsurface This method provides substantial enhancement offluorescence collection efficiency while rejecting backgroundfrom unbound fluors or impurities as it confines the flu-orescence detection volume to material within about onewavelength of the chip surface [121]

Generally the analyte is labeled by attaching a chro-mophore fluorophore or particle (dye containing semi-conductorquantum dot noble metal or scattering) to anantibody or nucleic acid strand that confers specific recogni-tion for optical detection Nanoparticles including quantumdots are finding increasing application Nanoparticle labelsconjugated with biomolecules have been used in a varietyof different assay application Nanoparticles offer adjustableand expandable reactive surface area compared to the moretraditional solid phase forms utilized in bioaffinity assays dueto the high surface-to-volume ratio Signal enhancement byconjugating nanoparticles with fluorescent luminescent andother measurable properties has enhanced detection limit byseveral foldsThe potential to multiplex along with the abilityto increase sensitivity and specificity without using enzymeshas increased the use of nanoparticles in immunoassays forearly detection It has been shown that using time resolvedfluorescence of lanthanides like europium nanoparticles withlong stokes shift can reduce background and increase detec-tion limit to as low as 05 pgmL of HIV p24 [122] The deter-mination of cancer biomarkers in serum and saliva usingquantum dot bioconjugate labels are used Quantum dotswere employed on chip for CD4+ T-cell counting in a POCapplication Aptamers were tethered to gold nanoparticles aspart of an LFA-like dry-reagent assay strip to detect thrombin[118] Thermal-lens microscopy (TLM) an alternative tofluorescence detection also uses dye labeling for detectionTLM detection integrated to a miniaturized ELISA devicealong with optical electronic and fluidic components coulddetect an LOD of 2 ngmL for total IgE measurement [123]


92 Electrochemical Detection

921 Electrochemical Immunosensors Electrochemical im-munoassays are the most commonly used analytical tech-niques for the quantitative detection of biomolecules fol-lowed by optical methods Electrochemical immunosensorsare not only sufficient to meet the demands for sensitiverapid and selective determination of analytes but can also beincorporated into robust portable andminiaturized devicesSpecifically the integration of electrochemical detection withmicrofluidic chips offers an attractive immunoassay platformwith significant advantages derived from the combinationof electrochemical analysis and a microfluidic system [124]In broad terms electrochemical immunosensors functionby detecting an electrical signal that arises from specificimmunoreactions that occur at the surface of an electrodeAccording to the type of electrical detection signal electro-chemical techniques are classified into three basic categoriesvoltammetry (current) potentiometry (potential shift) andimpedimetry (resistance) [125]

Some analytes are electroactive and can be measureddirectly without labeling electrochemical detection often uti-lizes tagging for analyte specificitywith either an electroactivespecies or an enzyme that converts an electrochemically silentspecies into electroactive one this approach also providessignal amplification of multiple orders of magnitude withdetection limits below 1 pM readily accessible [118 126] Ithas been shown that ultrasensitive capacitive immunosensoris capable of detecting subattogram per milliliter concentra-tions of p24 antigen [127]

922 Piezoelectric Immunosensors Piezoelectric devicesconvert a physical or mechanical change into electricalenergy and vice versa The commonest piezoelectric sensoris the quartz crystal microbalance (QCM) which exploitsthe change in the resonance of quartz crystals upon changesin their mass allowing binding of antigen to antibody (whenone of these is immobilised on the crystal surface) to bemeasured electrically [128]

923 Microcantilevers (MC) Sensors based on MCs arean attractive method for sensitive and label-free detectionOn the basis of advances in micro fabrication techniquesmassively parallel cantilevers can be facilitated inmicrofluidicdevices to independently monitor multiple targets [129] Twodistinctmodes ofMC immunosensors that are utilized for thesignal transduction of immune recognition binding events onthe MC surface are the deflection (cantilever bending) andresonance (resonant frequency shift) modes

Microcantilever-based devices were developed utilizingrotating resonance microcantilevers which measured thefrequency-shift of themicrocantilevermotion with respect tothe specific adsorbed mass to give sensors capable of mea-suring 120572-fetoprotein to less than 2 ngmL for the label-freePOC for early detection of hepatocellular carcinoma [130]Also a microring resonator immunosensor has been recentlydescribed which can detect multiple analytes (PSA 120572-fetoprotein CEA tumor necrosis factor-120572 and interleukin-8)

concurrently without loss of sensitivity or measurementprecision when compared to single-parameter analysis [131]

93 Magnetic Detection Magnetic particles are widely usedfor POC diagnostics because they can be used to precon-centrate and they can also be used as a labeling technologyfor detection without the requirements of fluors for 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 (MTJ) methods [132]

931 Label-Free Methods The label-free methods for POCdetection include SPR amperometric immunosensor andelectrochemical sensors based on mechanical transductionand direct electrochemical and optical transduction for ana-lytes possessing suitable characteristics

Silicon nanowire (SiNW) biosensors have been exten-sively studied in the last decade for the detection of biologicalmolecules as highly sensitive label-free and electrical toolsSiNW biosensors hold great promise to realize POC devicesfor disease diagnostics with potential for miniaturization andintegration [133]

Optical surface plasmon resonance (SPR) biosensorsrepresent themost advanced and developed optical label-freebiosensor technology Optical SPR biosensors are a powerfuldetection and analysis tool that have vast applications ininfectious disease diagnostics [134]

Mechanical transducers for POC applications oscillateor resonate including micro- and nanocantilevers [135] aswell as various acoustic wave devices such as the quartz-crystal microbalance (QCM) and a range of devices in thesurface acoustic wave family [136] Operating characteristicssuch as frequency and signal attenuation for piezoelec-tric devices or resistance and amplitude for piezoresistive(silicon) devices are affected by the mass and mechanicalproperties of molecules and materials linked to their oscil-lating surfaces like SPR they require only an immobilizedselective recognition layer Nonetheless ldquomass tagsrdquo denseparticles (typically Au) that bind selectively to the target cansignificantly enhance sensitivity The ldquobond rupture sensorsrdquouse acoustic energy to rupture bonds between immobilizedcapture antibodies and target microbes Because the energyand frequency of added energy can be adjusted nonspecif-ically and specifically captured particles can be desorbeddifferentially according to the bond strengths and massesproviding a unique discrimination mechanism [137]

932 Integrated Rapid-Diagnostic-Test Reader Platform ona Cell Phone Aydogan Ozcan group have demonstrated acell phone-based rapid-diagnostic-test (RDT) reader plat-form that can work with various lateral flow immuno-chromatographic assays and similar tests to sense the pres-ence of a target analyte in a sample This compact and cost-effective digital RDT reader weighing only 65 gmechanicallyattaches to the existing camera unit of a cell phone wherevarious types of RDTs can be inserted to be imaged in


reflection or transmissionmodes under light-emitting diode-(LED-) based illumination Captured raw images of thesetests are then digitally processed (within less than 02 secondsper image) through a smart application running on the cellphone for validation of the RDT as well as for automatedreading of its diagnostic result The same smart applicationthen transmits the resulting data together with the RDTimages and other related information (eg demographicdata) to a central server which presents the diagnosticresults on a world map through geotagging This dynamicspatiotemporal map of various RDT results can then beviewed and shared using internet browsers or through thesame cell phone application They have tested this platformon malaria tuberculosis (TB) and HIV RDTs by installing iton both Android-based smart phones and an iPhone [138]

933 Plasmonic ELISA Molly M Stevenrsquos group havedemonstrated an ultrasensitive ELISA which uses a signal-generation mechanism for biosensing that enables the detec-tion of a few molecules of analyte with the naked eyeThe enzyme label of an ELISA controls the growth of goldnanoparticles and generates colored solutions with distincttonality when the analyte is present HIV-1 capsid antigen p24was detected in whole serum at the ultralow concentrationof 1times10minus18 gmL of p24 and was also detected with the nakedeye in the sera of HIV-infected patients showing viral loadsundetectable by a gold standard nucleic acid-based test [139]

10 Opportunities and Challenges forCost-Efficient Implementation of NewPoint-of-Care Diagnostics for HIV

High-quality diagnostics are essential to fight against HIVand to reduce its transmission There are clear diagnosticneeds where conventional laboratory support is insufficientand not cost effective HIV rapid point-of-care (POC) testingfor initial HIV diagnosis has been successful but severalneeds remain Despite its clear advantages POC testinghas important limitations and laboratory-based testing willcontinue to be an important component of future diagnosticnetworks [140]

Manymiddle- and low-income countries implementationof rapid cost-effective POC HIV diagnostic screening havedeveloped algorithms for diagnosing HIV infection usingstrategic orthogonal testing assays without the need for addi-tional confirmatory testing [141] Use of saliva has broadenedthe testing but saliva samples have been associated withhigher false-positive rates in some testing sites [142] Despitethe advances and successes of HIV serological rapid assaysunmet gaps for qualitative POC applications remain Firstwe need to identify acute HIV infection because recentlyinfected individuals are seronegative but have extremelyhigh viral loads making them far more infectious comparedwith chronically infected individuals [143] Early detectioncan result in early treatment initiation which limits diseaseprogression and reduces transmission [144]

Secondwe need to diagnose infants born toHIV-infectedmothers particularly in sub-Saharan Africa Due to the

placental transfer of antibodies during pregnancy infantsmay test positive on serological assays yetmay not be infectedwith HIV [145] Currently infant diagnosis is primarilyperformed by collecting dried blood spots and transportingthe samples to central testing laboratories for DNA testing[146] However many infants go undiagnosed because oflong turnaround times or insufficient infrastructure (egunreliable sample transportation) [145]

Third we need appropriate tests for individuals vac-cinated with experimental HIV antigens in clinical tri-als [147 148] Vaccine volunteers mount specific immuneresponses to the vaccine constructs which react with manyserological diagnostic tests making future HIV diagnosisdifficult potentially unblinding trial staff and negativelyimpacting society [148] Similar assays may also be beneficialin resource-limited settings to monitor antiretroviral therapy(ART) effectiveness and the potential need to switch therapy[149] In each of these cases new rapid molecular POC HIVscreening tests will fill an important diagnostic gap

11 Future Trends

Strip-based tests with the use of low-cost polymer substrateslike paper and thread microfluidics and low-cost readerslike smart phones are also expected to facilitate home test-ing Digitaldroplet microfluidic devices are being activelyresearched and only recently have begun to enter the fieldof POC diagnostics will become popular The gap betweenldquotraditionalrdquo (eg porous nitrocellulose) lateral flow assaydevices and microfluidic platforms is narrowing with thedevelopment of these technologies While PCR remains agold standard for high sensitivity and specificity in mostcases there are other emerging related isothermal PCRtechniques to consider There are a range of related PCRtechniques such as RCA LAMP NASBA MDA TMASDA and LCA (rolling-circle amplification loop-mediatedisothermal amplification nucleic acid sequence-based ampli-fication multiple-displacement amplification transcription-mediated amplification strand-displacement amplificationand ligase chain reaction resp) as well as cleavase Invaderwhich have the potential to be used in POC settings Impor-tantly many of these methods are isothermal and operateat lower temperatures than PCR making their integrationwith microfluidic technologies more straightforward Fordetection new opticalmethods exploit phenomena includingupconversion high-brightness nanoparticles total-internal-reflection fluorescence (TIRF) SAF FRET and a range ofplasmon-based effects In many cases nanoparticles enhanceoptical detection The above technologies enhance signalreduce background or both combined with low-cost com-pact high-intensity solid-state light sources this drives POCLODs even lower Detectingmultiple analytes in a single POCtest is an important trend many of the most promising newPOC opportunities are in multianalyte tests or panels

12 Conclusion

This review has outlined some of the key developments inthe field of point-of-care technologies currently in use for


HIVAIDS as well as technologies that hold promise andcurrently under development for future use in improvingearly infant diagnosis and viral load monitoring particularlyin resource limited settings The timely development of thesetesting approaches will be critical to ushering in a newera of AIDS prevention with its goal of an ldquoAIDS-freerdquogenerationThe feasibility of achieving this goalwill be greatlyenhanced by implementing targeted improvements in testingand developing new therapies for HIVAIDS In recent yearsthere has been significant involvement of both private andpublic sectors in the development of diagnostic technologiesto meet the need for HIV testing in resource-limited settingsFuture strategies may utilize the most appropriate technolo-gies to help achieve prevention goals for HIVAIDS throughsustainable impact in the public health setting

Disclosure

The findings and conclusions in this paper have not beenformally disseminated by the Food and Drug Administrationand should not be construed to represent any Agency deter-mination or policy This review paper reflects the views ofthe authors and should not be construed to represent FDArsquosviews or policies Some productsmay not have been approvedby and are not endorsed by FDA for the usespotential usesdiscussed in the paper

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors wish to acknowledge Dr Krishna KumarDevadas and Dr Xue Wang for reviewing the paper

References

[1] K A S Pkowitz ldquoAIDSmdashthe first 20 yearsrdquo The New EnglandJournal of Medicine vol 344 no 23 pp 1764ndash1772 2001

[2] WHO UNICEF and UNAIDS ldquoEpidemic update and healthsector progress towards universal accessrdquo Progress ReportGlobal HIVAIDS Response WHO Geneva Switzerland 2011

[3] UNITAID ldquoHIVAIDS diagnostic technology landscaperdquo TechRep WHO Geneva Switzerland 2012

[4] R J Meagher A V Hatch R F Renzi and A K SinghldquoAn integrated microfluidic platform for sensitive and rapiddetection of biological toxinsrdquo Lab on a Chip vol 8 no 12 pp2046ndash2053 2008

[5] J Wang H Ahmad C Ma et al ldquoA self-powered one-step chipfor rapid quantitative and multiplexed detection of proteinsfrom pinpricks of whole bloodrdquo Lab on a Chip vol 10 no 22pp 3157ndash3162 2010

[6] C Jonsson M Aronsson G Rundstrom et al ldquoSilane-dextranchemistry on lateral flowpolymer chips for immunoassaysrdquoLabon a Chip vol 8 no 7 pp 1191ndash1197 2008

[7] M M Murtagh and T F Peter ldquoART 20 implications fordiagnostics in resource-limited settingsrdquo ClintonHealth AccessInitiative 2010

[8] G Wu and M H Zaman ldquoLow-cost tools for diagnosing andmonitoring HIV infection in low-resource settingsrdquo Bulletin ofthe WHO vol 90 pp 914ndash920 2012

[9] M R Parisi L Soldini G D Perri S Tiberi A Lazzarin and FB Lillo ldquoOffer of rapid testing and alternative biological samplesas practical tools to implement HIV screening programsrdquo NewMicrobiologica vol 32 no 4 pp 391ndash396 2009

[10] B M Branson ldquoThe future of HIV testingrdquo Journal of AcquiredImmune Deficiency Syndromes vol 55 supplement 2 pp S102ndashS105 2010

[11] G Beelaert and K Fransen ldquoEvaluation of a rapid and simplefourth-generationHIV screening assay for qualitative detectionof HIV p24 antigen andor antibodies to HIV-1 and HIV-2rdquoJournal of Virological Methods vol 168 no 1-2 pp 218ndash2222010

[12] J Fox H Dunn and S OrsquoShea ldquoLow rates of p24 antigendetection using a fourth-generation point of care HIV testrdquoSexually Transmitted Infections vol 87 no 2 pp 178ndash179 2011

[13] S Sivapalasingam S Essajee P N Nyambi et al ldquoHumanimmunodeficiency virus (HIV) reverse transcriptase activitycorrelates with HIV RNA load implications for resource-limited settingsrdquo Journal of Clinical Microbiology vol 43 no 8pp 3793ndash3796 2005

[14] K Steegen S Luchters NDeCabooter et al ldquoEvaluation of twocommercially available alternatives for HIV-1 viral load testingin resource-limited settingsrdquo Journal of VirologicalMethods vol146 no 1-2 pp 178ndash187 2007

[15] S Sivapalasingam B Wangechi F Marshed et al ldquoMonitoringvirologic responses to antiretroviral therapy in HIV-infectedadults in Kenya evaluation of a low-cost viral load assayrdquo PLoSOne vol 4 no 8 Article ID e6828 2009

[16] W Labbett A Garcia-Diaz Z Fox et al ldquoComparative evalua-tion of the ExaVir load version 3 reverse transcriptase assay formeasurement of human immunodeficiency virus type 1 plasmaloadrdquo Journal of Clinical Microbiology vol 47 no 10 pp 3266ndash3270 2009

[17] D Bonard F Rouet T A Toni et al ldquoField evaluation of animproved assay using a heat-dissociated p24 antigen for adultsmainly infected with HIV-1 CRF02 AG strains in Cote drsquoIvoireWestAfricardquo Journal of Acquired ImmuneDeficiency Syndromesvol 34 no 3 pp 267ndash273 2003

[18] V L Greengrass M M Plate P M Steele et al ldquoEvaluationof the Cavidi ExaVir Load assay (version 3) for plasma humanimmunodeficiency virus type 1 load monitoringrdquo Journal ofClinical Microbiology vol 47 no 9 pp 3011ndash3013 2009

[19] R Zachariah S D Reid P Chaillet M Massaquoi E JSchouten and A D Harries ldquoViewpoint why do we needa point-of-care CD4 test for low-income countriesrdquo TropicalMedicine and International Health vol 16 no 1 pp 37ndash41 2011

[20] W H A Chow C McCloskey Y Tong et al ldquoApplication ofisothermal helicase-dependent amplification with a disposabledetection device in a simple sensitive stool test for toxigenicClostridium difficilerdquo Journal of Molecular Diagnostics vol 10no 5 pp 452ndash458 2008

[21] T D Ly S Laperche C Brennan et al ldquoEvaluation of thesensitivity and specificity of six HIV combined p24 antigen andantibody assaysrdquo Journal of Virological Methods vol 122 no 2pp 185ndash194 2004


[22] T D Ly L Martin D Daghfal et al ldquoSeven human immun-odeficiency virus (HIV) antigen-antibody combination assaysevaluation ofHIV seroconversion sensitivity and subtype detec-tionrdquo Journal of Clinical Microbiology vol 39 no 9 pp 3122ndash3128 2001

[23] G Murphy and C Aitken ldquoHIV testing-the perspective fromacross the pondrdquo Journal of Clinical Virology vol 52 supple-ment 1 pp S71ndashS76 2011

[24] B Weber ldquoScreening of HIV infection role of molecular andimmunological assaysrdquo Expert Review of Molecular Diagnosticsvol 6 no 3 pp 399ndash411 2006

[25] BWeber L Gurtler RThorstensson et al ldquoMulticenter evalua-tion of a new automated fourth-generation human immunode-ficiency virus screening assay with a sensitive antigen detectionmodule and high specificityrdquo Journal of Clinical Microbiologyvol 40 no 6 pp 1938ndash1946 2002

[26] T D Ly A Ebel V Faucher V Fihman and S Laperche ldquoCouldthe newHIV combined p24 antigen and antibody assays replacep24 antigen specific assaysrdquo Journal of Virological Methods vol143 no 1 pp 86ndash94 2007

[27] S P Layne M J Merges M Dembo et al ldquoFactors underlyingspontaneous inactivation and susceptibility to neutralization ofhuman immunodeficiency virusrdquo Virology vol 189 no 2 pp695ndash714 1992

[28] C Bentsen L McLaughlin E Mitchell et al ldquoPerformanceevaluation of the Bio-Rad Laboratories GS HIV Combo AgAbEIA a 4th generation HIV assay for the simultaneous detectionof HIV p24 antigen and antibodies to HIV-1 (groups M andO) and HIV-2 in human serum or plasmardquo Journal of ClinicalVirology vol 52 supplement 1 pp S57ndashS61 2011

[29] P Chavez L Wesolowski P Patel K Delaney and S M OwenldquoEvaluation of the performance of the Abbott ARCHITECTHIV AgAb combo assayrdquo Journal of Clinical Virology vol 52supplement 1 pp S51ndashS55 2011

[30] M W Pandori J Hackett Jr B Louie et al ldquoAssessment ofthe ability of a fourth-generation immunoassay for humanimmunodeficiency virus (HIV) antibody and p24 antigen todetect both acute and recent HIV infections in a high-risksettingrdquo Journal of ClinicalMicrobiology vol 47 no 8 pp 2639ndash2642 2009

[31] L Laursen ldquoPoint-of-care tests poised to alter course of HIVtreatmentrdquo Nature Medicine vol 18 article 1156 2012

[32] A Cachafeiro G G Sherman A H Sohn C Beck-Sagueand S A Fiscus ldquoDiagnosis of human immunodeficiencyvirus type 1 infection in infants by use of dried blood spotsand an ultrasensitive p24 antigen assayrdquo Journal of ClinicalMicrobiology vol 47 no 2 pp 459ndash462 2009

[33] S Workman S K Wells C-P Pau et al ldquoRapid detection ofHIV-1 p24 antigen using magnetic immuno-chromatography(MICT)rdquo Journal of VirologicalMethods vol 160 no 1-2 pp 14ndash21 2009

[34] Z A Parpia R Elghanian A Nabatiyan D R Hardie andD M Kelso ldquoP24 antigen rapid test for diagnosis of acutepediatricHIV infectionrdquo Journal of Acquired ImmuneDeficiencySyndromes vol 55 no 4 pp 413ndash419 2010

[35] M M Murtagh ldquoHIVAIDS diagnostics landscaperdquo UNITAIDTechnical Report WHO Geneva Switzerland 2011

[36] S Mtapuri-Zinyowera M Chideme D Mangwanya et alldquoEvaluation of the PIMA point-of-care CD4 analyzer in VCTclinics in Zimbabwerdquo Journal of Acquired Immune DeficiencySyndromes vol 55 no 1 pp 1ndash7 2010

[37] N Sitoe E LueckeN Tembe et al ldquoAbsolute and percentCD4+T-cell enumeration by flow cytometry using capillary bloodrdquoJournal of ImmunologicalMethods vol 372 no 1-2 pp 1ndash6 2011

[38] Y C Manabe Y Wang A Elbireer B Auerbach and B Castel-nuovo ldquoEvaluation of portable point-of-care CD4 counter withhigh sensitivity for detecting patients eligible for antiretroviraltherapyrdquo PLoS One vol 7 no 4 Article ID e34319 2012

[39] D K Glencross L M Coetzee M Faal et al ldquoPerformanceevaluation of the Pima point-of-care CD4 analyser using cap-illary blood sampling in field tests in South Africardquo Journal ofthe International AIDS Society vol 15 no 1 article 3 2012

[40] S Tanriverdi L Chen and S Chen ldquoA rapid and automatedsample-to-resultHIV load test for near-patient applicationrdquoTheJournal of Infectious Diseases vol 201 supplement 1 pp S52ndashS58 2010

[41] R L Hodinka T Nagashunmugam and D Malamud ldquoDetec-tion of human immunodeficiency virus antibodies in oralfluidsrdquo Clinical and Diagnostic Laboratory Immunology vol 5no 4 pp 419ndash426 1998

[42] W A OrsquoBrien P M Hartigan D Martin et al ldquoChanges inplasma HIV-1 RNA and CD4+ lymphocyte counts and the riskof progression to AIDSrdquoThe New England Journal of Medicinevol 334 no 7 pp 426ndash431 1996

[43] J A Sterne M May D Costagliola et al ldquoTiming of initiationof antiretroviral therapy in AIDS-free HIV-1-infected patientsa collaborative analysis of 18 HIV cohort studiesrdquo The Lancetvol 373 no 9672 pp 1352ndash1363 2009

[44] WHO ldquoAntiretroviral therapy for HIV infection in adults andadolescents recommendations for a public health approachrdquo2010 Revision WHO Geneva Switzerland 2010

[45] ldquoReview of CD4 technologiesrdquo GS Presentation GenevaWorld Health Organization 2011 httpwwwwhointhivtopicstreatment

[46] I V Jani N E Sitoe P L Chongo et al ldquoAccurate CD4 T-cell enumeration and antiretroviral drug toxicity monitoring inprimary healthcare clinics using point-of-care testingrdquo Journalof the International AIDS Society vol 25 no 6 pp 807ndash812 2011

[47] X Cheng D Irimia M Dixon et al ldquoA microchip approachfor practical label-free CD4+ T-cell counting of HIV-infectedsubjects in resource-poor settingsrdquo Journal of Acquired ImmuneDeficiency Syndromes vol 45 no 3 pp 257ndash261 2007

[48] X Cheng Y-S Liu D Irimia et al ldquoCell detection and countingthrough cell lysate impedance spectroscopy in microfluidicdevicesrdquo Lab on a Chip vol 7 no 6 pp 746ndash755 2007

[49] D S Boyle K R Hawkins M S Steele M Singhal andX Cheng ldquoEmerging technologies for point-of-care CD4 T-lymphocyte countingrdquo Trends in Biotechnology vol 30 no 1pp 45ndash54 2012

[50] C Willyard ldquoSimpler tests for immune cells could transformAIDS care in Africardquo Nature Medicine vol 13 no 10 p 11312007

[51] D L Robertson J P Anderson J A Bradac et al ldquoHIV-1nomenclature proposalrdquo Science vol 288 no 5463 pp 55ndash562000

[52] ldquoHIV sequence databaserdquo 2012 httpwwwhivlanlgovcont-entsequenceHIVCRFsCRFshtml

[53] WHO UNICEF and UNAIDS ldquoTowards universal accessscaling up priority HIVAIDS interventions in the healthsectorrdquo Progress Report WHO Geneva Switzerland 2009

[54] S Wang F Xu and U Demirci ldquoAdvances in developing HIV-1 viral load assays for resource-limited settingsrdquo BiotechnologyAdvances vol 28 no 6 pp 770ndash781 2010


[55] P A Volberding and S G Deeks ldquoAntiretroviral therapy andmanagement of HIV infectionrdquo The Lancet vol 376 no 9734pp 49ndash62 2010

[56] D M Moore A Awor R Downing et al ldquoCD4+ T-cell countmonitoring does not accurately identify HIV-infected adultswith virologie failure receiving antiretroviral therapyrdquo Journal ofAcquired Immune Deficiency Syndromes vol 49 no 5 pp 477ndash484 2008

[57] B M Branson ldquoState of the art for diagnosis of HIV infectionrdquoClinical Infectious Diseases vol 45 supplement 4 pp S221ndashS225 2007

[58] D A Anderson S M Crowe and M Garcia ldquoPoint-of-caretestingrdquo Current HIVAIDS Reports vol 8 no 1 pp 31ndash37 2011

[59] S Butto B Suligoi E Fanales-Belasio and M RaimondoldquoLaboratory diagnostics for HIV infectionrdquo Annali dellrsquoIstitutoSuperiore di Sanita vol 46 no 1 pp 24ndash33 2010

[60] M Vincent Y Xu and H Kong ldquoHelicase-dependent isother-mal DNA amplificationrdquo EuropeanMolecular Biology Organiza-tion Reports vol 5 no 8 pp 795ndash800 2004

[61] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-moleculecounting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[62] J Van Ness L K Van Ness and D J Galas ldquoIsothermalreactions for the amplification of oligonucleotidesrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 100 no 8 pp 4504ndash4509 2003

[63] K A Curtis D L Rudolph and S M Owen ldquoRapid detectionof HIV-1 by reverse-transcription loop-mediated isothermalamplification (RT-LAMP)rdquo Journal of Virological Methods vol151 no 2 pp 264ndash270 2008

[64] K A Curtis D L Rudolph and SMOwen ldquoSequence-specificdetection method for reverse transcription loop-mediatedisothermal amplification of HIV-1rdquo Journal of Medical Virologyvol 81 no 6 pp 966ndash972 2009

[65] K A Curtis D L Rudolph I Nejad et al ldquoIsothermalamplification using a chemical heating device for point-of-caredetection of HIV-1rdquo PLoS One vol 7 no 2 Article ID e314322012

[66] Y Mori T Hirano and T Notomi ldquoSequence specific visualdetection of LAMP reactions by addition of cationic polymersrdquoBMC Biotechnology vol 6 article 3 2006

[67] P Francois M Tangomo J Hibbs et al ldquoRobustness of aloop-mediated isothermal amplification reaction for diagnosticapplicationsrdquo FEMS Immunology andMedicalMicrobiology vol62 no 1 pp 41ndash48 2011

[68] C Liu E Geva M Mauk et al ldquoAn isothermal amplificationreactor with an integrated isolationmembrane for point-of-caredetection of infectious diseasesrdquo Analyst vol 136 no 10 pp2069ndash2076 2011

[69] M Goto E Honda A Ogura A Nomoto and K-I HanakildquoColorimetric detection of loop-mediated isothermal amplifi-cation reaction by using hydroxy naphthol bluerdquo BioTechniquesvol 46 no 3 pp 167ndash172 2009

[70] B Deiman P Van Aarle and P Sillekens ldquoCharacteristicsand applications of nucleic acid sequence-based amplification(NASBA)rdquo Applied Biochemistry and Biotechnology Part B vol20 no 2 pp 163ndash179 2002

[71] J Jean D H DrsquoSouza and L-A Jaykus ldquoMultiplex nucleicacid sequence-based amplification for simultaneous detectionof several enteric viruses in model ready-to-eat foodsrdquo Applied

and Environmental Microbiology vol 70 no 11 pp 6603ndash66102004

[72] O Piepenburg C HWilliams D L Stemple and N A ArmesldquoDNA detection using recombination proteinsrdquo PLoS Biologyvol 4 article e204 no 7 2006

[73] T Notomi H Okayama H Masubuchi et al ldquoLoop-mediatedisothermal amplification of DNArdquo Nucleic Acids Research vol28 article E63 no 12 2000

[74] Y Kimura M J L De Hoon S Aoki et al ldquoOptimization ofturn-back primers in isothermal amplificationrdquo Nucleic AcidsResearch vol 39 article e59 no 9 2011

[75] K Nagamine T Hase and T Notomi ldquoAccelerated reactionby loop-mediated isothermal amplification using loop primersrdquoMolecular and Cellular Probes vol 16 no 3 pp 223ndash229 2002

[76] Y Mori K Nagamine N Tomita and T Notomi ldquoDetectionof loop-mediated isothermal amplification reaction by turbidityderived frommagnesium pyrophosphate formationrdquo Biochemi-cal and Biophysical Research Communications vol 289 no 1 pp150ndash154 2001

[77] L L M Poon B W Y Wong E H T Ma et al ldquoSensitive andinexpensive molecular test for falciparum malaria defectingPlasmodium falciparum DNA directly from heat-treated bloodby loop-mediated isothermal amplificationrdquoClinical Chemistryvol 52 no 2 pp 303ndash306 2006

[78] Y Mori M Kitao N Tomita and T Notomi ldquoReal-timeturbidimetry of LAMP reaction for quantifying template DNArdquoJournal of Biochemical and Biophysical Methods vol 59 no 2pp 145ndash157 2004

[79] KNagamine KWatanabe KOhtsuka THase andTNotomildquoLoop-mediated isothermal amplification reaction using a non-denatured templaterdquo Clinical Chemistry vol 47 no 9 pp 1742ndash1743 2001

[80] R Suzuki M Ihira Y Enomoto et al ldquoHeat denaturationincreases the sensitivity of the cytomegalovirus loop-mediatedisothermal amplification methodrdquo Microbiology and Immunol-ogy vol 54 no 8 pp 466ndash470 2010

[81] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[82] G Kumar E Garnova M Reagin and A Vidali ldquoImprovedmultiple displacement amplification with 12059329DNApolymerasefor genotyping of single human cellsrdquoBioTechniques vol 44 no7 pp 879ndash890 2008

[83] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[84] S Henriksson A-L Blomstrom L Fuxler C Fossum MBerg and M Nilsson ldquoDevelopment of an in situ assay forsimultaneous detection of the genomic and replicative form ofPCV2 using padlock probes and rolling circle amplificationrdquoVirology Journal vol 8 article 37 2011

[85] J Melin J Jarvius J Goransson and M Nilsson ldquoHomoge-neous amplified single-molecule detection characterization ofkey parametersrdquo Analytical Biochemistry vol 368 no 2 pp230ndash238 2007

[86] P M Williams R Li N A Johnson G Wright J-D Heathand R D Gascoyne ldquoA novel method of amplification ofFFPET-derived RNA enables accurate disease classificationwith microarraysrdquo Journal of Molecular Diagnostics vol 12 no5 pp 680ndash686 2010


[87] I Biswas and P Hsieh ldquoIdentification and characterization of athermostable MutS homolog fromThermus aquaticusrdquo Journalof Biological Chemistry vol 271 no 9 pp 5040ndash5048 1996

[88] Y Mitani A Lezhava Y Kawai et al ldquoRapid SNP diagnos-tics using asymmetric isothermal amplification and a newmismatch-suppression technologyrdquo Nature Methods vol 4 no3 pp 257ndash262 2007

[89] Y Kimura A Oguchi-Katayama Y Kawai et al ldquoTail variationof the folding primer affects the SmartAmp2 process differ-entlyrdquo Biochemical and Biophysical Research Communicationsvol 383 no 4 pp 455ndash459 2009

[90] G T Walker M C Little J G Nadeau and D D ShankldquoIsothermal in vitro amplification of DNA by a restrictionenzymeDNA polymerase systemrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 89 no1 pp 392ndash396 1992

[91] G T Walker M S Fraiser J L Schram M C LittleJ G Nadeau and D P Malinowski ldquoStrand displacementamplificationmdashan isothermal in vitro DNA amplification tech-niquerdquoNucleic Acids Research vol 20 no 7 pp 1691ndash1696 1992

[92] H Jia Z Li C Liu and Y Cheng ldquoUltrasensitive detectionof microRNAs by exponential isothermal amplificationrdquo Ange-wandte Chemie vol 49 no 32 pp 5498ndash5501 2010

[93] M Shimada F Hino H Sagawa H Mukai K Asada and IKato ldquoDevelopment of the detection system forMycobacteriumtuberculosis DNA by using the isothermal DNA amplificationmethod ICANrdquo Rinsho Byori vol 50 no 5 pp 528ndash532 2002

[94] H Mukai T Uemori O Takeda et al ldquoHighly efficient isother-malDNAamplification systemusing three elements of 51015840-DNA-RNA-31015840 chimeric primers RNaseH and strand-displacing DNApolymeraserdquo Journal of Biochemistry vol 142 no 2 pp 273ndash2812007

[95] T Uemori H Mukai O Takeda et al ldquoInvestigation of themolecular mechanism of ICAN a novel gene amplificationmethodrdquo Journal of Biochemistry vol 142 no 2 pp 283ndash2922007

[96] L Kulinsky Z Noroozi and M Madou ldquoPresent technologyand future trends in point-of-care microfluidic diagnosticsrdquoMethods in Molecular Biology vol 949 pp 3ndash23 2013

[97] P F Mens A van Amerongen P Sawa P A Kager andH D F H Schallig ldquoMolecular diagnosis of malaria in thefield development of a novel 1-step nucleic acid lateral flowimmunoassay for the detection of all 4 human Plasmodium sppand its evaluation inMbita KenyardquoDiagnosticMicrobiology andInfectious Disease vol 61 no 4 pp 421ndash427 2008

[98] M Blazkova M Koets P Rauch and A van AmerongenldquoDevelopment of a nucleic acid lateral flow immunoassay forsimultaneous detection of listeria spp and listeria monocyto-genes in foodrdquoEuropean FoodResearch andTechnology vol 229no 6 pp 867ndash874 2009

[99] S Assadollahi C Reininger R Palkovits P Pointl and TSchalkhammer ldquoFrom lateral flowdevices to a novel nano-colormicrofluidic assayrdquo Sensors vol 9 no 8 pp 6084ndash6100 2009

[100] W R Seitz ldquoImmunoassay labels based on chemiluminescenceand bioluminescencerdquo Clinical Biochemistry vol 17 no 2 pp120ndash125 1984

[101] P Corstjens M Zuiderwijk A Brink et al ldquoUse of up-converting phosphor reporters in lateral-flow assays to detectspecific nucleic acid sequences a rapid sensitive DNA testto identify human papillomavirus type 16 infectionrdquo ClinicalChemistry vol 47 no 10 pp 1885ndash1893 2001

[102] X Chu X Fu K Chen G-L Shen and R-Q Yu ldquoAnelectrochemical strippingmetalloimmunoassay based on silver-enhanced gold nanoparticle labelrdquo Biosensors and Bioelectron-ics vol 20 no 9 pp 1805ndash1812 2005

[103] P Von Lode ldquoPoint-of-care immunotesting approaching theanalytical performance of central laboratory methodsrdquo ClinicalBiochemistry vol 38 no 7 pp 591ndash606 2005

[104] A Kupstat M U Kumke and N Hildebrandt ldquoTowardsensitive quantitative point-of-care testing (POCT) of proteinmarkers miniaturization of a homogeneous time-resolvedfluoroimmunoassay for prostate-specific antigen detectionrdquoAnalyst vol 136 no 5 pp 1029ndash1035 2011

[105] B A Rohrman V Leautaud E Molyneux and R R Richards-Kortum ldquoA lateral flow assay for quantitative detection ofamplifiedHIV-1 RNArdquo PLoS One vol 7 Article ID e45611 2012

[106] P Craw and W Balachandran ldquoIsothermal nucleic acid ampli-fication technologies for point-of-care diagnostics a criticalreviewrdquo Lab on a Chip vol 12 no 14 pp 2469ndash2486 2012

[107] F Vitzthum and J Bernhagen ldquoSYBR Green I an ultrasensitivefluorescent dye for double-standed DNA quantification insolution and other applicationsrdquo Recent Research Developmentsin Analytical Biochemistry vol 2 pp 65ndash93 2002

[108] P M Holland R D Abramson R Watson and D H GelfandldquoDetection of specific polymerase chain reaction product byutilizing the 51015840ndash31015840 exonuclease activity of Thermus aquaticusDNA polymeraserdquo Proceedings of the National Academy ofSciences of the United States of America vol 88 no 16 pp 7276ndash7280 1991

[109] AWMartinez S T Phillips GMWhitesides and E CarrilholdquoDiagnostics for the developing world microfluidic paper-based analytical devicesrdquoAnalytical Chemistry vol 82 no 1 pp3ndash10 2010

[110] W Zhao M A Brook and Y Li ldquoDesign of gold nanoparticle-based colorimetric biosensing assaysrdquoChemBioChem vol 9 no15 pp 2363ndash2371 2008

[111] R Koonakosit ldquoSemiquantitative determination of urinaryglucose comparison of home-made strip and routine testsrdquoJournal of the Medical Association of Thailand vol 83 no 1 ppS152ndashS160 2000

[112] D D Liana B Raguse J J Gooding and E Chow ldquoRecentadvances in paper-based sensorsrdquo Sensors vol 12 no 9 pp11505ndash11526 2012

[113] Y Adiguzel and H Kulah ldquoCMOS cell sensors for point-of-carediagnosticsrdquo Sensors vol 12 no 8 pp 10042ndash10066 2012

[114] K-H Lee S-H Choi J-O Lee M-J Sohn J-B Yoon andG-H Cho ldquoAn autonomous CMOS hysteretic sensor for thedetection of desorption-free DNA hybridizationrdquo Biosensorsand Bioelectronics vol 26 no 11 pp 4591ndash4595 2011

[115] J M Rothberg W Hinz T M Rearick et al ldquoAn integratedsemiconductor device enabling non-optical genome sequenc-ingrdquo Nature vol 475 no 7356 pp 348ndash352 2011

[116] SWangM Esfahani U A Gurkan F Inci D R Kuritzkes andU Demirci ldquoEfficient on-chip isolation of HIV subtypesrdquo Labon a Chip vol 12 no 8 pp 1508ndash1515 2012

[117] S Schumacher J Nestler T Otto et al ldquoHighly-integrated lab-on-chip system for point-of-care multiparameter analysisrdquo Labon a Chip vol 12 no 3 pp 464ndash473 2012

[118] VGubala L FHarris A J RiccoMX Tan andD EWilliamsldquoPoint of care diagnostics status and futurerdquo Analytical Chem-istry vol 84 no 2 pp 487ndash515 2012


[119] L Gervais and E Delamarche ldquoToward one-step point-of-careimmunodiagnostics using capillary-driven microfluidics andPDMS substratesrdquo Lab on a Chip vol 9 no 23 pp 3330ndash33372009

[120] S Fournier-Bidoz T L Jennings J M Klostranec et al ldquoFacileand rapid one-stepmass preparation of quantum-dot barcodesrdquoAngewandte Chemie vol 47 no 30 pp 5577ndash5581 2008

[121] T Ruckstuhl C M Winterflood and S Seeger ldquoSupercriticalangle fluorescence immunoassay platformrdquo Analytical Chem-istry vol 83 no 6 pp 2345ndash2350 2011

[122] S Tang and I Hewlett ldquoNanoparticle-based immunoassays forsensitive and early detection of HIV-1 capsid (p24) antigenrdquoTheJournal of Infectious Diseases vol 201 supplement 1 pp S59ndashS64 2010

[123] T Ohashi KMawatari K SatoM Tokeshi and T Kitamori ldquoAmicro-ELISA system for the rapid and sensitivemeasurement oftotal and specific immunoglobulin E and clinical application toallergy diagnosisrdquo Lab on a Chip vol 9 no 7 pp 991ndash995 2009

[124] M Hervas M A Lopez and A Escarpa ldquoElectrochemicalimmunosensing on board microfluidic chip platformsrdquo Trendsin Analytical Chemistry vol 31 pp 109ndash128 2012

[125] D W Kimmel G Leblanc M E Meschievitz and D ECliffel ldquoElectrochemical sensors and biosensorsrdquo AnalyticalChemistry vol 84 no 2 pp 685ndash707 2012

[126] C Li L A Shutter P-M Wu C H Ahn and R K NarayanldquoPotential of a simple lab-on-a-tube for point-of-care measure-ments of multiple analytesrdquo Lab on a Chip vol 10 no 11 pp1476ndash1479 2010

[127] K Teeparuksapun M Hedstrom E Y Wong S Tang I KHewlett and B Mattiasson ldquoUltrasensitive detection of HIV-1 p24 antigen using nanofunctionalized surfaces in a capacitiveimmunosensorrdquoAnalytical Chemistry vol 82 no 20 pp 8406ndash8411 2010

[128] K N Han C A Li and G H Seong ldquoMicrofluidic chips forimmunoassaysrdquo Analytical Chemistry vol 6 pp 119ndash141 2013

[129] R McKendry J Zhang Y Arntz et al ldquoMultiple label-free biodetection and quantitative DNA-binding assays on ananomechanical cantilever arrayrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no15 pp 9783ndash9788 2002

[130] Y Liu X Li Z Zhang G Zuo Z Cheng and H YuldquoNanogram per milliliter-level immunologic detection ofalpha-fetoprotein with integrated rotating-resonance micro-cantilevers for early-stage diagnosis of heptocellular carci-nomardquo Biomedical Microdevices vol 11 no 1 pp 183ndash191 2009

[131] A LWashburn M S Luchansky A L Bowman and R C Bai-ley ldquoQuantitative label-free detection of five protein biomark-ers using multiplexed arrays of silicon photonic microringresonatorsrdquoAnalytical Chemistry vol 82 no 1 pp 69ndash72 2010

[132] S X Wang and A M Taratorin Magnetic Information StorageTechnology Electromagnetism Academic Press 1st edition1999

[133] G J Zhang and Y Ning ldquoSilicon nanowire biosensor and itsapplications in disease diagnostics a reviewrdquoAnalytica ChimicaActa vol 749 pp 1ndash15 2012

[134] XGuo ldquoSurface plasmon resonance based biosensor techniquea reviewrdquo Journal of Biophotonics vol 5 no 7 pp 483ndash501 2012

[135] P S Waggoner and H G Craighead ldquoMicromdashand nanome-chanical sensors for environmental chemical and biologicaldetectionrdquo Lab on a Chip vol 7 no 10 pp 1238ndash1255 2007

[136] M-I Rocha-Gaso CMarch-Iborra AMontoya-Baides andAArnau-Vives ldquoSurface generated acoustic wave biosensors forthe detection of pathogens a reviewrdquo Sensors vol 9 no 7 pp5740ndash5769 2009

[137] E R Hirst Y J Yuan W L Xu and J E Bronlund ldquoBond-rupture immunosensorsmdasha reviewrdquo Biosensors and Bioelectron-ics vol 23 no 12 pp 1759ndash1768 2008

[138] O Mudanyali S Dimitrov U Sikora S Padmanabhan INavruz and A Ozcan ldquoIntegrated rapid-diagnostic-test readerplatform on a cellphonerdquo Lab on a Chip vol 12 no 15 pp 2678ndash2686 2012

[139] R de la Rica and M M Stevens ldquoPlasmonic ELISA for theultrasensitive detection of disease biomarkers with the nakedeyerdquo Nature Nanotechnology vol 7 no 12 pp 821ndash824 2012

[140] M Schito T F Peter S Cavanaugh et al ldquoOpportunities andchallenges for cost-efficient implementation of new point-of-care diagnostics for HIV and tuberculosisrdquo Journal of InfectiousDiseases vol 205 supplement 2 pp S169ndashS180 2012

[141] T Crucitti D Taylor G Beelaert K Fransen and L VanDamme ldquoPerformance of a rapid and simple HIV testingalgorithm in a multicenter phase III microbicide clinical trialrdquoClinical and Vaccine Immunology vol 18 no 9 pp 1480ndash14852011

[142] S J Reynolds and JMuwonga ldquoOraQuickAdvance RapidHIV-12 antibody testrdquo Expert Review of Molecular Diagnostics vol4 no 5 pp 587ndash591 2004

[143] K A Powers C Poole A E Pettifor and M S CohenldquoRethinking the heterosexual infectivity of HIV-1 a systematicreview andmeta-analysisrdquoTheLancet Infectious Diseases vol 8no 9 pp 553ndash563 2008

[144] C D Pilcher L Eaton S Kalichman C Bisol and R Da SilvaDe Souza ldquoApproaching ldquoHIV eliminationrdquo interventions foracute HIV infectionrdquo Current HIVAIDS Reports vol 3 no 4pp 160ndash168 2006

[145] T L Creek G G Sherman J Nkengasong et al ldquoInfant humanimmunodeficiency virus diagnosis in resource-limited settingsissues technologies and country experiencesrdquo American Jour-nal of Obstetrics and Gynecology vol 197 no 3 pp S64ndashS712007

[146] B H Chohan S Emery D Wamalwa et al ldquoEvaluation of asingle round polymerase chain reaction assay using dried bloodspots for diagnosis of HIV-1 infection in infants in an Africansettingrdquo BMC Pediatrics vol 11 article 18 2011

[147] E Van Braeckel M Koutsoukos P Bourguignon F Clement LMcNally andG Leroux-Roels ldquoVaccine-inducedHIV seropos-itivity a problem on the riserdquo Journal of Clinical Virology vol50 no 4 pp 334ndash337 2011

[148] M L Schito M P DrsquoSouza S M Owen and M P BuschldquoChallenges for rapid molecular HIV diagnosticsrdquo The Journalof Infectious Diseases vol 201 supplement 1 pp S1ndashS6 2010

[149] F Rouet and C Rouzioux ldquoThe measurement of HIV-1 viralload in resource-limited settings how and whererdquo ClinicalLaboratory vol 53 no 3-4 pp 135ndash148 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


are already in place and well established However portablequantitative tests for rapid POC HIV monitoring have onlyrecently been introduced to the market

There is great need for initial diagnosis staging andongoing monitoring of HIV Among those that presentthe most persistent challenges to improved access andefficiency are CD4 viral load and early infant diagnosis(EID) The great majority of testing options available todayare laboratory-based platforms performed 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 for patients in periurbanand rural settings The success of high-quality antiretroviraltherapy (ART) depends on simple affordable reliable andquality-assured POC diagnostics for use in resource limitedsettings (RLS) POCdiagnostics canmakeARTmore scalableand will allow ART service delivery to be significantlydecentralized to the community level At the same timesimplifying diagnostic technologies that could potentiallyreduce the cost of diagnosing and monitoring patients livingwith HIVAIDS without diminishing the quality of care isimportant for treatment success The World Health Orga-nization (WHO) has set criteria for at or near POC usemdashknown as ldquoASSUREDrdquo criteria meaning that they are (or willbe) affordable sensitive specific user-friendly robustrapidequipment-free and deliverable to those who need the test[3]

POCdiagnostics are in vitrodiagnostics (IVD) that donotinvolve the use of laboratory staff and facilities to provide theresultThe analytical ldquotargetsrdquo include proteins nucleic acidsmetabolites drugs dissolved ions and gases human cells andmicrobes Test samples can be blood saliva urine or otherbodily fluids or (semi-) solids Whether used ldquonear patientrdquoin a hospital clinic or doctorrsquos office or administered at hometo maintain health manage disease or monitor therapy or inthe field to test the safety of water food or compliance withlaws and regulations these tests accept a sample with little orno preparation and provide a result the ldquoanswerrdquo in secondsto hours [4]

The 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 of coloron a strip of paper or polymer increasingly however readersranging from hand-held devices to bench-top instrumentsread the analytical test provide a comprehensible resultand if necessary control and operate the sample-containingplatform that executes the analytical process These devicesshould be capable of using nanoliters to milliliters of complexbiological media with femtomolar to millimolar concentra-tions of analytes [5] The devices should be inexpensivedisposable units or cartridges that include microfluidic fea-tures to provide or control sample preparation flow ratemixing with reagents reaction time associated with bindingevents filtration of nonanalytical components of the sampleseparation of interfering agents and of multiple analytes andan effective measurement capability [6]

ldquoWhy POC Diagnostics for HIVrdquo POC measurementsprovide results rapidly where needed and often with major

time savings samples do not travel to a laboratory to awaitthe attention of 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 for accuracy or reliability

2 HIV Diagnostics

Diagnostics for HIVAIDS can generally be divided intothree test categories (i) tests to facilitate initial diagnosis(ii) tests to stage the patient and (iii) tests to monitor thepatient both before and after initiation of ART There aregenerally accepted algorithms and tests used at each stage ofthe infection [7]

21 Low-Cost Diagnostic Tools for Monitoring HIV Low-costtechnologies to diagnose andmonitorHIV infection in devel-oping countries are a major subject of current research inhealth care settings in the developing world With increasingneed to provide access to affordable HIVmonitoring servicesin rural areas of developing countries much work has beenfocused on the development of point-of-care technologiesthat are affordable robust easy to use and portable and ofsufficient quantitative accuracy to enable clinical decisionmaking For diagnosis of HIV infection some low-cost testssuch as lateral flow tests and enzyme-linked immunosorbentassays are already in place and well established Howeverportable quantitative tests for rapid HIV monitoring at thepoint of care have only recently been introduced to themarket[8] The Ora Quick Rapid HIV-12 Antibody Test is a lateralflow rapid test for oral fluid specimens that perform as well asblood-based tests even at low concentrations ofHIV antigensin oral fluid [9] The Aware HIV-12 U with 972 sensitivityand 100 specificity 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 p24 antigen aswell as tests for early infant diagnosis and for HIV-1 andHIV-2 subtype differentiation [10] Detection of both antibody andantigen during the acute phase of infection is particularlybeneficial to prevention efforts because the rate of HIVtransmission is several folds higher during this phase and canaccount for up to 50 of newHIV infectionsThe DetermineHIV-12 AgAb Combo is an immunochromatographic rapidtest that detects antigen and antibody separately In twoindependent studies the Determine HIV-12 AgAb Comboshowed sensitivities of 50 and 86 for antigen detection[11 12]

22 HIV Viral Load Monitoring HIV viral load monitoringis crucial for therapy and staging of the disease Reverse tran-scriptase (RT) an enzyme present during viral replicationcorrelates linearly with HIV load and CD4+ T-cell count [13]Measuring this RT protein may be an alternative for resourcelimited settings as its yielding results in a few hours and costs80 less than nucleic acid tests [14]

























































































10copiesmL




















9 Detection





























11 Future Trends


12 Conclusion




Disclosure




Acknowledgments


References
































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































































































10copiesmL




















9 Detection





























11 Future Trends


12 Conclusion




Disclosure




Acknowledgments


References
































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























































































10copiesmL




















9 Detection





























11 Future Trends


12 Conclusion




Disclosure




Acknowledgments


References
































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































































10copiesmL




















9 Detection





























11 Future Trends


12 Conclusion




Disclosure




Acknowledgments


References
































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of










































































10copiesmL




















9 Detection





























11 Future Trends


12 Conclusion




Disclosure




Acknowledgments


References
































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































































10copiesmL




















9 Detection





























11 Future Trends


12 Conclusion




Disclosure




Acknowledgments


References
































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















































10copiesmL




















9 Detection





























11 Future Trends


12 Conclusion




Disclosure




Acknowledgments


References
































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of









































10copiesmL




















9 Detection





























11 Future Trends


12 Conclusion




Disclosure




Acknowledgments


References
































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































10copiesmL




















9 Detection





























11 Future Trends


12 Conclusion




Disclosure




Acknowledgments


References
































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























10copiesmL




















9 Detection





























11 Future Trends


12 Conclusion




Disclosure




Acknowledgments


References
































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























9 Detection





























11 Future Trends


12 Conclusion




Disclosure




Acknowledgments


References
































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















9 Detection





























11 Future Trends


12 Conclusion




Disclosure




Acknowledgments


References
































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































11 Future Trends


12 Conclusion




Disclosure




Acknowledgments


References
































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























11 Future Trends


12 Conclusion




Disclosure




Acknowledgments


References
































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Disclosure




Acknowledgments


References
































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Review Article Point of Care Technologies for HIVdownloads.hindawi.com/journals/art/2014/497046.pdf · 2019. 7. 31. · ongoing monitoring of HIV. Among those that present the most