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Journal of Clinical Virology 52S (2011) S71– S76

Contents lists available at SciVerse ScienceDirect

Journal of Clinical Virology

j ourna l ho mepage: www.elsev ier .com/ locate / j cv

IV testing—The perspective from across the pond�

ary Murphya,∗, Celia Aitkenb

Microbiology Services, Health Protection Agency, 61 Colindale Avenue, Colindale, London NW9 5HT, UKWest of Scotland Specialist Virology Centre, Gartnavel General Hospital, 1053 Great Western Road, Glasgow G12 0YN, UK

r t i c l e i n f o

eywords:IVesting algorithms

a b s t r a c t

The first assay to screen blood donations for HIV was licensed in the USA in 1985. Since then there hasbeen a dramatic increase in the types and numbers of assays available for HIV testing coupled withimprovements in the sensitivity and specificity of these assays. However, with this increase in choice thealgorithms for the initial diagnosis and confirmation of HIV infection have also increased in diversity andcomplexity and no uniform algorithm exists.

Different regulatory regimes have meant that different assays and assay formats are availableworldwide. In the UK we have been fortunate in having access to the so called 4th generation HIV anti-gen/antibody assays for 10 years. The first 4th generation assay in the US was licensed last year. Theavailability of this class of assays has led to the development of new algorithms for use in the US marketand this paper describes how after many years of diversity the HIV algorithms between the UK and USare now converging.

The publication of the CLSI (Clinical and Laboratory Standardsnstitute) document M53-A: Criteria for Laboratory Testing andiagnosis of Human Immunodeficiency Virus Infection: Approveduideline provides extensive up to date guidance on laboratoryethods and their interpretation in HIV infection.1

The evidence for the recommendations has been gathered overhe last five or so years by both the Association of Public Health Lab-ratories (APHL) and the Centers for Disease Control and PreventionCDC, Atlanta, GA).2 Regulatory constraints e.g. delays in approvalf the 4th generation diagnostic assays have led to a greater diver-ence between the laboratory procedures in the United KingdomUK) and United States (US), and while there have been revisionso the testing guidelines the diagnostic algorithm in the US hasemained largely unchanged since 1989. Following FDA approvalor the 4th generation assays, it is likely that over the next few yearshere will be a greater convergence of the 2 countries’ approacheso HIV diagnosis. Even within Europe there are different approacheso testing and confirmation as shown by Devaux et al.3 (Table 1).ocal guidelines/algorithms have largely been adapted as newevelopments have occurred with many institutions implementing

hanges ahead of any local/national guidance. Although in the USuidelines are generally set by the CDC in collaboration with otherrganizations, in the UK it is not always clear where the official

� Except where cited the views are those of the authors.∗ Corresponding author.

E-mail addresses: gary.murphy@hpa.org.uk (G. Murphy),elia.aitken@ggc.scot.nhs.uk (C. Aitken).

386-6532/$ – see front matter © 2011 Published by Elsevier B.V.oi:10.1016/j.jcv.2011.09.027

© 2011 Published by Elsevier B.V.

recommendation should come from. Several organizations includ-ing the Department of Health, Health Protection Agency (HPA), andHIV organizations such as The British HIV Association (BHIVA), allproduce guidance.

HIV services in the UK are provided both in teaching hospi-tals and district general hospitals. The laboratory work supportingthese services are performed either in a dedicated virology ormicrobiology laboratory. Most dedicated virology services arebased in teaching hospitals, and in addition to providing virol-ogy services for their own patients they will also provide areference service for local microbiology laboratories. The HPAis an independent UK organization established in 2003, whoseprimary remit is to “protect the public against health hazardsand infectious diseases”, in Scotland this is devolved to HPS(Health Protection Scotland). HPA Colindale is responsible forspecialist infectious diseases surveillance, including blood borneviruses, it advises at both the local and national level and isresponsible for the writing and dissemination of the NationalStandard Methods and Algorithms. These algorithms are regularlyreviewed and updated as required. They offer laboratories acrossthe country a minimum standard to follow, however they are notmandatory.

This article will focus on HIV testing from the UK perspective; itwill not deal with screening for organ or blood donors.

1. Historical perspective

Ever since 1985, HIV screening assays have relied principallyon the detection of infection through demonstrating the presence

S72 G. Murphy, C. Aitken / Journal of Clinic

Table 1HIV testing algorithms used in the countries in the WHO European Region, 2006.

Screening test Confirmation test No. of countries

ELISA No test 22nd ELISA 17Western blot 34Immunoblot 13Other 52nd + 3rd ELISA or other 4

PCR}

10p24 antigenViral culture

Source: Devaux I, Alix J, Likatavicius G, Herida M, Nielsen S, Hamers FF,et al. Human immunodeficiency virus (HIV) and acquired immunodeficiencysyndrome (AIDS) case reporting in the World Health Organization Euro-ph

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compared to the only marginal gain in sensitivity. In the US routine

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ean Region in 2006. Euro Surveill 2008;13(39), pii 18988. Available online:ttp://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=18988.

f anti-HIV antibodies in the serum of the subject under test. Thearly assays (1st and 2nd generation) detected IgG only with theain difference being the source of the viral antigen—1st genera-

ion assays relied on crude or purified viral lysate from HIV-1 only,hereas the 2nd generation assays used either synthetic oligopep-

ides or carefully selected antigens produced by DNA technologyo produce selected viral antigens from both HIV-1 and HIV-2. Thest generation assays lacked sensitivity and specificity4 and wereot able to detect the antibody response to the different HIV-1lades. Further refinements led to the 3rd generation assays which

ffered the additional advantage of detecting HIV-1/2 IgM and IgG.n average third generation assays were able to detect HIV-1/2ntibodies 9–15 days after the first detection of HIV p24 antigenFig. 1). In the UK the introduction of fourth generation HIV-1/2

Fig. 1. Improvement in seneproduced from: Perry KR, Ramskill S, Eglin RP, Barbara JA, Parry JV. Improvement in th

al Virology 52S (2011) S71– S76

assays in the National Blood Service and most hospital laborato-ries has reduced this period even further. This is achieved by thesimultaneous detection of p24 antigen and HIV-1/2 antibodies ina single ELISA.5 The superiority of these assays has been recentlyreviewed by Perry et al.6 and is shown in Fig. 1. In the United Statesthe first 4th generation assay was licensed for use in 2010. This wasa major step forward because previously some US laboratories stillrelied on the relatively insensitive 2nd generation anti-HIV assayswhile others had switched to the limited number of 3rd generationHIV antibody assays available. The recommendation of the originalCDC/APHL HIV testing algorithm required all anti-HIV repeatedlyreactive samples to be confirmed by Western blot. This algorithmbecame outdated once the sensitivity and specificity of the primaryscreening test surpassed those of the confirmatory assay. The lim-ited but growing access to the newer generation of assays becamea driver of change for US testing algorithms. However, to counterthe insensitivity of their antibody screening programmes there wassome, off label, use of RNA testing of anti-HIV negative specimensin certain populations such as STI clinic attendees where acute HIVinfection was likely. To make this process cost effective specimenswere pooled for testing. The presence of high viral load presentin very early infection means that specimens can be diluted manytimes, typically 40–90 specimens per pool, without greatly affect-ing the sensitivity of the assay.7 In the UK because of the presenceof 4th generation assays and a standalone p24 antigen assay the useof pooled RNA testing has not been recommended in the diagnosticenvironment due to the increased risk of false positive results when

viral load testing is not approved for primary screening and diag-nosis although one assay, the Gen-Probe Aptima RNA qualitativeassay, is licensed for the confirmation of a reactive EIA. The use of

sitivity of HIV assays.e performance of HIV screening kits. Transfus Med 2008;18:228–40.

Clinical Virology 52S (2011) S71– S76 S73

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Table 2Factors affecting choice of HIV algorithm.

Factor Reason

HIV prevalence, HIV incidence,prevailing subtype, etc.

In a high prevalence, high incidencearea you may want to use an assay thatis targeted to identify acute infections.If your population has a diversesubtype present you would want tochoose an algorithm that detects thissubtype

Testing volumes, automatedplatforms

A low throughput laboratory may notbe able to justify the cost of automatedplatforms and thus have to choosedifferent assays

Local/national regulationsaccess to assays

Is the appropriate assay licensed foruse in a particular country/state?

Local contracts Do local purchasing contracts limit youto assays from one supplier?

Ease of access to patient followup/access to care

Is the patient likely to be lost to followup if a rapid result not obtained, will apatient be able to access care based onthe outcome of their test result?

Staff skills, experience andknowledge

Do the staff have the skill andexperience to run a full confirmatoryHIV service or will they refer reactivesto a secondary centre?

Algorithm purpose, specimentypes

Is the algorithm to be used fordiagnostic testing or populationsurveillance? What specimen type willbe available—serum/plasma, driedblood spot, saliva?

Cost Is it cost efficient to perform testing,

G. Murphy, C. Aitken / Journal of

ooled RNA testing has, however, been implemented nationally inhe UK since 2007 for the screening of blood donations and is alsosed in the US.

. Factors influencing the need to change algorithms

Using the definition of an algorithm as “a step by step prob-em solving procedure using a defined number of steps” it is clearhat no single algorithm can cover the requirements of screen-ng/testing and confirmation for HIV in all settings and patientroups. Consideration also needs to be given of how, if possible,o incorporate the newer assays (e.g. 4th generation assays anducleic acid amplification tests [NAT] testing) into current algo-ithms or to the development of new algorithms. The improvementn sensitivity of the newer screening assays means that previ-usly accepted confirmatory tests, such as Western blot, may nowail to confirm specimens from individuals who are truly infectedue to their relative insensitivity to the screening assay. Similarlyhere are now a number of assays using alternatives to blood sam-les (e.g. oral fluid and dried blood spots) which also need to be

ncluded in the diagnostic algorithms. Hence as the sensitivity andange of assays have increased, the complexity of the confirma-ory algorithms has increased rather than decreased and has alsoontributed to the differences in HIV diagnostic algorithms seenorldwide.

In addition to the changes seen in technology there have alsoeen significant changes in the clinical management of HIV dis-ase, coupled with a growing awareness that a number of HIV casesere being missed. A recent audit in 2006, by the British HIV Asso-

iation (BHIVA) showed that 24% of deaths were a direct result of aate diagnosis (CD4 < 200).8 These late diagnoses were in part due tohe missed opportunities to test individuals when they visited theiramily doctor for other reasons but also reflected a lack of aware-ess, particularly in sexually active gay men, around the need foregular testing. This was supported by national surveillance dataonfirming that around one third of individuals with HIV werenaware of their diagnosis,9 and in the US it was shown that 50%f transmissions were from people unaware of their infection.10 As

result in the UK it became a political as well as a clinical imper-tive to “normalize” HIV testing and offer it in a variety of clinicalettings with guidance being issued by the Chief Medical Officersf both England and Scotland. BHIVA produced “Guidelines for HIVesting 2008”.11 These guidelines included the following recom-

endations on whom and how to test:

. Who to testAll patients with a recognisable risk factor e.g. sexual expo-

sure (either lifestyle, or contact with person from country ofhigh prevalence), intravenous drug use, or presence of a clinicalindicator disease.

Screening of all patients attending antenatal clinic, termina-tion clinics, sexual health clinics, people from high prevalencecountries, and people registering with a general practitioner oracute medical admissions where the prevalence is >2 per 1000.

Other groups include those who have symptoms that maybe related to primary HIV infection and neonatal samples frombabies born to HIV infected mothers.

. How to testThe recommended first-line assay should be one that tests for

HIV antibody and antigen (4th generation), with results available

within 72 h.

Confirmation requires 3 independent assays, which shouldhave two 4th generation assays and at least one assay distin-guishing between HIV-1 and HIV.

and if so to what level, or shouldspecimen be referred elsewhere?

3. Factors affecting choice of algorithm

There are many factors (laboratory and clinical) affecting thechoice both of assays (screening and confirmation) and howthey are incorporated into an algorithm (Table 2). In the UK,population size, local prevalence and workload are importantconsiderations in determining type of screening assay (manualor automated system) and whether confirmation will be offeredlocally or not. Most laboratories will also need to consider theperformance of tests both for general screening and urgent test-ing as most services will offer screening of antenatal attendersas part of national policy and testing of patients considered tobe at risk of infection such as sexual health clinic attenders. Cur-rently available automated platforms and assay formats allowsame day testing to be performed where the laboratory is closeto the clinic and an urgent HIV result can often be obtained in2–4 h. Where such platforms are not available, where special-ized laboratory facilities are not available or when a particularlyurgent screening result is required, such as during labour whenthe antenatal screen result is missing or in STI clinics, there hasbeen an increase in the uptake of point of care and rapid HIVtesting although a laboratory confirmation of infection is stillrequired.

Laboratory services in the UK are currently under review, andthis is likely to affect both the size and skill mix of most laborato-ries. Whereas virology centres will have the expertise to performscreening for HIV and confirmation, this may not be the case for allmicrobiology laboratories, in these cases they will need to be ableto refer their samples onto specialist centres. This will undoubtedlyimpact on turn around times.

4. The algorithms

The HPA have produced an algorithm for HIV screening (Fig. 2)but no algorithm is approved for HIV confirmation and this is left to

S74 G. Murphy, C. Aitken / Journal of Clinical Virology 52S (2011) S71– S76

HIV scS lgoriths

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Fig. 2. UK national

ource: Health Protection Agency. Anti-HIV screening—minimum testing atandardmethods.org.uk/pdf sops.asp.

he individual laboratory.14 An example of a possible confirmatory

lgorithm is shown in Fig. 3, it is important that the local clinicaleam understand at what stage a sample is classified as confirmed.

ithin the UK the accepted screening test is a 4th generation assayn blood. No nationally approved algorithm exists for screening

Fig. 3. Example of a possible dual 4th generatio

reening algorithm.m. National Standard Method VSOP 11 Issue 2; 2009, http://www.hpa-

with other specimen matrices or with rapid tests. This is in

contrast to the algorithms produced by CLSI which describe 6different algorithms describing the type of assay used for theinitial screen and covering different sample types such as oralfluid.

n HIV screening/confirmatory algorithm.

G. Murphy, C. Aitken / Journal of Clinical Virology 52S (2011) S71– S76 S75

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In the UK, on identification of a reactive specimen, repeat test-ng of this sample with either the same test or one of equivalentensitivity is recommended. At this point full confirmation with anlternate assay should then be performed either in the same lab-ratory or referred elsewhere (Fig. 3). As discrepant results mayndicate very early infection, the algorithms direct the laboratoryither to requesting a further sample 1–2 weeks later or NAT detec-ion.

The uptake of 4th generation assays in the United States hased to a proposed new testing algorithm (Fig. 4) which now showsreater concordance with the algorithms currently in use in the UKor laboratory confirmation.

A recent audit (2010) of diagnostic tests in the UK revealedhat the bulk of HIV testing is being carried out in non special-st virology centres, i.e. microbiology facilities (only 35 of 122 labsurveyed were designated Clinical Virology Centres, the remainderere virology units or microbiology laboratories). Nearly all the

aboratories surveyed (90%) test antenatal samples in addition toamples from other patient groups. 87% of laboratories test >5000amples/year with 20% doing more than 20,000 tests/year. Despiteuidance, 3 out of the 122 laboratories surveyed still use a 3rdeneration assay for screening. Of those laboratories performingonfirmation (n = 51) only 33 distinguished between HIV 1 and 2the remainder sent samples for testing elsewhere) and in 5 of themn insufficient number or combination of confirmatory assays weresed.12 The Western blot as part of a confirmatory algorithm is onlysed in the 2 Scottish reference laboratories, where it is used as anxtra test in addition to the 3 assays used for confirmation.

. Other considerations

In the UK confirmation of patient identity is also critical; this ischieved by testing the original sample from the clot and request-ng a second sample. Further samples are also requested when theerological profile is unusual, or when acute infection is suspected.n the latter situation most laboratories would perform NAT testinghemselves (either locally or refer the sample to a specialist cen-re). However it should be remembered these commercial viral load

ssays are not licensed for this use, although, one of these assaysoes have a licence for use as a confirmatory test in the US. Falseositives with very low viral loads have occasionally been reported

n this situation.

IV testing algorithm.

As stated there are no national algorithms in the UK for the useof rapid/near patient testing or for alternative specimen types toblood. However, rapid/near patient testing is being piloted acrossa number of different health care settings, and are used in someconfirmatory algorithms within local laboratories. Preliminary datasupport the use of these devices providing there is the appropriatesupport for patients and the necessary quality control.13 Currentlytheir use in non hospital settings is increasing but there is not anydata on how widespread this is. Within laboratories the audit men-tioned above12 has shown that 24 out of 133 laboratories use rapidtesting devices, with 9 laboratories using 3rd generation assays (6of which have incorporated it into their confirmatory algorithm)and 15 laboratories use a 4th generation assay (5 of which haveincorporated the 4th generation RTD as part of their laboratories’standard confirmatory algorithm rather than just as a screeningassay).

In addition to HIV diagnosis in England and Wales the HealthProtection Agency has introduced a programme to test every newHIV diagnosis for evidence of recent HIV infection. A similar pro-gram is starting in Scotland. This program uses avidity testing todistinguish between recent (last 3 months) and past infection, andis the only program that also returns the result to the patient (Scot-land has still to decide whether to anonymize the program or not).The data can be compared to the New HIV Diagnosis database andwill allow the estimation of HIV incidence in different populations.Identifying patients with recent infection also allows for targetedcontact tracing thereby identifying those individuals at greatest riskwhen the index was at their most infectious. Other countries suchas France and the United States also have programmes for identify-ing recent infection but these use different laboratory techniquesand anonymized specimens.

6. Conclusions

Since the discovery of HIV there have been tremendousadvances in the care of HIV infected patients. Despite theseadvances it has been difficult to have common algorithms for diag-nosis and confirmation of HIV infection. Although the sensitivity

and range of HIV-1/2 diagnostic assays have improved significantly,delays in regulatory approval of the newer assays have complicatedthe establishment of testing algorithms. Following FDA approval of4th generation assays in 2010, it is likely that there will be fewer

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76 G. Murphy, C. Aitken / Journal of

ifferences between the algorithms in the US and UK, with mostifferences being in the detail, reflecting local clinical need.

unding

None.

ompeting interests

No financial disclosures were reported by the authors of thisaper.

thical approval

Not required.

eferences

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al Virology 52S (2011) S71– S76

Region in 2006. Euro Surveill 2008;13(39), pii 18988. Available online:http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=18988.

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