- Email: [email protected]
HIV testing—The perspective from across the pond
Journal of Clinical Virology 52S (2011) S71–S76
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
HIV testing—The perspective from across the pond夽 Gary Murphy a,∗ , Celia Aitken b a b
Microbiology Services, Health Protection Agency, 61 Colindale Avenue, Colindale, London NW9 5HT, UK West of Scotland Specialist Virology Centre, Gartnavel General Hospital, 1053 Great Western Road, Glasgow G12 0YN, UK
a r t i c l e Keywords: HIV Testing algorithms
i n f o
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 has been a dramatic increase in the types and numbers of assays available for HIV testing coupled with improvements in the sensitivity and specificity of these assays. However, with this increase in choice the algorithms for the initial diagnosis and confirmation of HIV infection have also increased in diversity and complexity and no uniform algorithm exists. Different regulatory regimes have meant that different assays and assay formats are available worldwide. In the UK we have been fortunate in having access to the so called 4th generation HIV antigen/antibody assays for 10 years. The first 4th generation assay in the US was licensed last year. The availability of this class of assays has led to the development of new algorithms for use in the US market and this paper describes how after many years of diversity the HIV algorithms between the UK and US are now converging. © 2011 Published by Elsevier B.V.
The publication of the CLSI (Clinical and Laboratory Standards Institute) document M53-A: Criteria for Laboratory Testing and Diagnosis of Human Immunodeficiency Virus Infection: Approved Guideline provides extensive up to date guidance on laboratory methods and their interpretation in HIV infection.1 The evidence for the recommendations has been gathered over the last five or so years by both the Association of Public Health Laboratories (APHL) and the Centers for Disease Control and Prevention (CDC, Atlanta, GA).2 Regulatory constraints e.g. delays in approval of the 4th generation diagnostic assays have led to a greater divergence between the laboratory procedures in the United Kingdom (UK) and United States (US), and while there have been revisions to the testing guidelines the diagnostic algorithm in the US has remained largely unchanged since 1989. Following FDA approval for the 4th generation assays, it is likely that over the next few years there will be a greater convergence of the 2 countries’ approaches to HIV diagnosis. Even within Europe there are different approaches to testing and confirmation as shown by Devaux et al.3 (Table 1). Local guidelines/algorithms have largely been adapted as new developments have occurred with many institutions implementing changes ahead of any local/national guidance. Although in the US guidelines are generally set by the CDC in collaboration with other organizations, in the UK it is not always clear where the official
夽 Except where cited the views are those of the authors. ∗ Corresponding author. E-mail addresses: [email protected] (G. Murphy), [email protected] (C. Aitken). 1386-6532/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.jcv.2011.09.027
recommendation should come from. Several organizations including the Department of Health, Health Protection Agency (HPA), and HIV organizations such as The British HIV Association (BHIVA), all produce guidance. HIV services in the UK are provided both in teaching hospitals and district general hospitals. The laboratory work supporting these services are performed either in a dedicated virology or microbiology laboratory. Most dedicated virology services are based in teaching hospitals, and in addition to providing virology services for their own patients they will also provide a reference service for local microbiology laboratories. The HPA is an independent UK organization established in 2003, whose primary remit is to “protect the public against health hazards and infectious diseases”, in Scotland this is devolved to HPS (Health Protection Scotland). HPA Colindale is responsible for specialist infectious diseases surveillance, including blood borne viruses, it advises at both the local and national level and is responsible for the writing and dissemination of the National Standard Methods and Algorithms. These algorithms are regularly reviewed and updated as required. They offer laboratories across the country a minimum standard to follow, however they are not mandatory. This article will focus on HIV testing from the UK perspective; it will not deal with screening for organ or blood donors. 1. Historical perspective Ever since 1985, HIV screening assays have relied principally on the detection of infection through demonstrating the presence
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Table 1 HIV testing algorithms used in the countries in the WHO European Region, 2006. Screening test
Confirmation test
No. of countries
ELISA
No test 2nd ELISA Western blot Immunoblot Other 2nd + 3rd ELISA or other
2 17 34 13 5 4
PCR p24 antigen Viral culture
10
Source: Devaux I, Alix J, Likatavicius G, Herida M, Nielsen S, Hamers FF, et al. Human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) case reporting in the World Health Organization European Region in 2006. Euro Surveill 2008;13(39), pii 18988. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=18988.
of anti-HIV antibodies in the serum of the subject under test. The early assays (1st and 2nd generation) detected IgG only with the main difference being the source of the viral antigen—1st generation assays relied on crude or purified viral lysate from HIV-1 only, whereas the 2nd generation assays used either synthetic oligopeptides or carefully selected antigens produced by DNA technology to produce selected viral antigens from both HIV-1 and HIV-2. The 1st generation assays lacked sensitivity and specificity4 and were not able to detect the antibody response to the different HIV-1 clades. Further refinements led to the 3rd generation assays which offered the additional advantage of detecting HIV-1/2 IgM and IgG. On average third generation assays were able to detect HIV-1/2 antibodies 9–15 days after the first detection of HIV p24 antigen (Fig. 1). In the UK the introduction of fourth generation HIV-1/2
assays in the National Blood Service and most hospital laboratories has reduced this period even further. This is achieved by the simultaneous detection of p24 antigen and HIV-1/2 antibodies in a single ELISA.5 The superiority of these assays has been recently reviewed by Perry et al.6 and is shown in Fig. 1. In the United States the first 4th generation assay was licensed for use in 2010. This was a major step forward because previously some US laboratories still relied on the relatively insensitive 2nd generation anti-HIV assays while others had switched to the limited number of 3rd generation HIV antibody assays available. The recommendation of the original CDC/APHL HIV testing algorithm required all anti-HIV repeatedly reactive samples to be confirmed by Western blot. This algorithm became outdated once the sensitivity and specificity of the primary screening test surpassed those of the confirmatory assay. The limited but growing access to the newer generation of assays became a driver of change for US testing algorithms. However, to counter the insensitivity of their antibody screening programmes there was some, off label, use of RNA testing of anti-HIV negative specimens in certain populations such as STI clinic attendees where acute HIV infection was likely. To make this process cost effective specimens were pooled for testing. The presence of high viral load present in very early infection means that specimens can be diluted many times, typically 40–90 specimens per pool, without greatly affecting the sensitivity of the assay.7 In the UK because of the presence of 4th generation assays and a standalone p24 antigen assay the use of pooled RNA testing has not been recommended in the diagnostic environment due to the increased risk of false positive results when compared to the only marginal gain in sensitivity. In the US routine viral load testing is not approved for primary screening and diagnosis although one assay, the Gen-Probe Aptima RNA qualitative assay, is licensed for the confirmation of a reactive EIA. The use of
Fig. 1. Improvement in sensitivity of HIV assays. Reproduced from: Perry KR, Ramskill S, Eglin RP, Barbara JA, Parry JV. Improvement in the performance of HIV screening kits. Transfus Med 2008;18:228–40.
G. Murphy, C. Aitken / Journal of Clinical Virology 52S (2011) S71–S76
pooled RNA testing has, however, been implemented nationally in the UK since 2007 for the screening of blood donations and is also used in the US.
Table 2 Factors affecting choice of HIV algorithm. Factor
Reason
HIV prevalence, HIV incidence, prevailing subtype, etc.
In a high prevalence, high incidence area you may want to use an assay that is targeted to identify acute infections. If your population has a diverse subtype present you would want to choose an algorithm that detects this subtype A low throughput laboratory may not be able to justify the cost of automated platforms and thus have to choose different assays Is the appropriate assay licensed for use in a particular country/state? Do local purchasing contracts limit you to assays from one supplier? Is the patient likely to be lost to follow up if a rapid result not obtained, will a patient be able to access care based on the outcome of their test result? Do the staff have the skill and experience to run a full confirmatory HIV service or will they refer reactives to a secondary centre? Is the algorithm to be used for diagnostic testing or population surveillance? What specimen type will be available—serum/plasma, dried blood spot, saliva? Is it cost efficient to perform testing, and if so to what level, or should specimen be referred elsewhere?
2. Factors influencing the need to change algorithms Using the definition of an algorithm as “a step by step problem solving procedure using a defined number of steps” it is clear that no single algorithm can cover the requirements of screening/testing and confirmation for HIV in all settings and patient groups. Consideration also needs to be given of how, if possible, to incorporate the newer assays (e.g. 4th generation assays and nucleic acid amplification tests [NAT] testing) into current algorithms or to the development of new algorithms. The improvement in sensitivity of the newer screening assays means that previously accepted confirmatory tests, such as Western blot, may now fail to confirm specimens from individuals who are truly infected due to their relative insensitivity to the screening assay. Similarly there are now a number of assays using alternatives to blood samples (e.g. oral fluid and dried blood spots) which also need to be included in the diagnostic algorithms. Hence as the sensitivity and range of assays have increased, the complexity of the confirmatory algorithms has increased rather than decreased and has also contributed to the differences in HIV diagnostic algorithms seen worldwide. In addition to the changes seen in technology there have also been significant changes in the clinical management of HIV disease, coupled with a growing awareness that a number of HIV cases were being missed. A recent audit in 2006, by the British HIV Association (BHIVA) showed that 24% of deaths were a direct result of a late diagnosis (CD4 < 200).8 These late diagnoses were in part due to the missed opportunities to test individuals when they visited their family doctor for other reasons but also reflected a lack of awareness, particularly in sexually active gay men, around the need for regular testing. This was supported by national surveillance data confirming that around one third of individuals with HIV were unaware of their diagnosis,9 and in the US it was shown that 50% of transmissions were from people unaware of their infection.10 As a result in the UK it became a political as well as a clinical imperative to “normalize” HIV testing and offer it in a variety of clinical settings with guidance being issued by the Chief Medical Officers of both England and Scotland. BHIVA produced “Guidelines for HIV testing 2008”.11 These guidelines included the following recommendations on whom and how to test:
1. Who to test All patients with a recognisable risk factor e.g. sexual exposure (either lifestyle, or contact with person from country of high prevalence), intravenous drug use, or presence of a clinical indicator disease. Screening of all patients attending antenatal clinic, termination clinics, sexual health clinics, people from high prevalence countries, and people registering with a general practitioner or acute medical admissions where the prevalence is >2 per 1000. Other groups include those who have symptoms that may be related to primary HIV infection and neonatal samples from babies born to HIV infected mothers. 2. How to test The 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 should have two 4th generation assays and at least one assay distinguishing between HIV-1 and HIV.
S73
Testing volumes, automated platforms
Local/national regulations access to assays Local contracts Ease of access to patient follow up/access to care
Staff skills, experience and knowledge
Algorithm purpose, specimen types
Cost
3. Factors affecting choice of algorithm There are many factors (laboratory and clinical) affecting the choice both of assays (screening and confirmation) and how they are incorporated into an algorithm (Table 2). In the UK, population size, local prevalence and workload are important considerations in determining type of screening assay (manual or automated system) and whether confirmation will be offered locally or not. Most laboratories will also need to consider the performance of tests both for general screening and urgent testing as most services will offer screening of antenatal attenders as part of national policy and testing of patients considered to be at risk of infection such as sexual health clinic attenders. Currently available automated platforms and assay formats allow same day testing to be performed where the laboratory is close to the clinic and an urgent HIV result can often be obtained in 2–4 h. Where such platforms are not available, where specialized laboratory facilities are not available or when a particularly urgent screening result is required, such as during labour when the antenatal screen result is missing or in STI clinics, there has been an increase in the uptake of point of care and rapid HIV testing although a laboratory confirmation of infection is still required. Laboratory services in the UK are currently under review, and this is likely to affect both the size and skill mix of most laboratories. Whereas virology centres will have the expertise to perform screening for HIV and confirmation, this may not be the case for all microbiology laboratories, in these cases they will need to be able to refer their samples onto specialist centres. This will undoubtedly impact 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
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Fig. 2. UK national HIV screening algorithm. Source: Health Protection Agency. Anti-HIV screening—minimum testing algorithm. National Standard Method VSOP 11 Issue 2; 2009, http://www.hpastandardmethods.org.uk/pdf sops.asp.
the individual laboratory.14 An example of a possible confirmatory algorithm is shown in Fig. 3, it is important that the local clinical team understand at what stage a sample is classified as confirmed. Within the UK the accepted screening test is a 4th generation assay on blood. No nationally approved algorithm exists for screening
with other specimen matrices or with rapid tests. This is in contrast to the algorithms produced by CLSI which describe 6 different algorithms describing the type of assay used for the initial screen and covering different sample types such as oral fluid.
Fig. 3. Example of a possible dual 4th generation HIV screening/confirmatory algorithm.
G. Murphy, C. Aitken / Journal of Clinical Virology 52S (2011) S71–S76
S75
Fig. 4. Proposed unified US HIV testing algorithm.
In the UK, on identification of a reactive specimen, repeat testing of this sample with either the same test or one of equivalent sensitivity is recommended. At this point full confirmation with an alternate assay should then be performed either in the same laboratory or referred elsewhere (Fig. 3). As discrepant results may indicate very early infection, the algorithms direct the laboratory either to requesting a further sample 1–2 weeks later or NAT detection. The uptake of 4th generation assays in the United States has led to a proposed new testing algorithm (Fig. 4) which now shows greater concordance with the algorithms currently in use in the UK for laboratory confirmation. A recent audit (2010) of diagnostic tests in the UK revealed that the bulk of HIV testing is being carried out in non specialist virology centres, i.e. microbiology facilities (only 35 of 122 labs surveyed were designated Clinical Virology Centres, the remainder were virology units or microbiology laboratories). Nearly all the laboratories surveyed (90%) test antenatal samples in addition to samples from other patient groups. 87% of laboratories test >5000 samples/year with 20% doing more than 20,000 tests/year. Despite guidance, 3 out of the 122 laboratories surveyed still use a 3rd generation assay for screening. Of those laboratories performing confirmation (n = 51) only 33 distinguished between HIV 1 and 2 (the remainder sent samples for testing elsewhere) and in 5 of them an insufficient number or combination of confirmatory assays were used.12 The Western blot as part of a confirmatory algorithm is only used in the 2 Scottish reference laboratories, where it is used as an extra test in addition to the 3 assays used for confirmation. 5. Other considerations In the UK confirmation of patient identity is also critical; this is achieved by testing the original sample from the clot and requesting a second sample. Further samples are also requested when the serological profile is unusual, or when acute infection is suspected. In the latter situation most laboratories would perform NAT testing themselves (either locally or refer the sample to a specialist centre). However it should be remembered these commercial viral load assays are not licensed for this use, although, one of these assays does have a licence for use as a confirmatory test in the US. False positives with very low viral loads have occasionally been reported in this situation.
As stated there are no national algorithms in the UK for the use of rapid/near patient testing or for alternative specimen types to blood. However, rapid/near patient testing is being piloted across a number of different health care settings, and are used in some confirmatory algorithms within local laboratories. Preliminary data support the use of these devices providing there is the appropriate support for patients and the necessary quality control.13 Currently their use in non hospital settings is increasing but there is not any data on how widespread this is. Within laboratories the audit mentioned above12 has shown that 24 out of 133 laboratories use rapid testing devices, with 9 laboratories using 3rd generation assays (6 of which have incorporated it into their confirmatory algorithm) and 15 laboratories use a 4th generation assay (5 of which have incorporated the 4th generation RTD as part of their laboratories’ standard confirmatory algorithm rather than just as a screening assay). In addition to HIV diagnosis in England and Wales the Health Protection Agency has introduced a programme to test every new HIV diagnosis for evidence of recent HIV infection. A similar program is starting in Scotland. This program uses avidity testing to distinguish between recent (last 3 months) and past infection, and is the only program that also returns the result to the patient (Scotland has still to decide whether to anonymize the program or not). The data can be compared to the New HIV Diagnosis database and will allow the estimation of HIV incidence in different populations. Identifying patients with recent infection also allows for targeted contact tracing thereby identifying those individuals at greatest risk when the index was at their most infectious. Other countries such as France and the United States also have programmes for identifying recent infection but these use different laboratory techniques and anonymized specimens. 6. Conclusions Since the discovery of HIV there have been tremendous advances in the care of HIV infected patients. Despite these advances it has been difficult to have common algorithms for diagnosis 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 complicated the establishment of testing algorithms. Following FDA approval of 4th generation assays in 2010, it is likely that there will be fewer
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differences between the algorithms in the US and UK, with most differences being in the detail, reflecting local clinical need.
4.
Funding
5.
None.
6.
Competing interests No financial disclosures were reported by the authors of this paper.
7.
8. 9.
Ethical approval Not required. References 1. Clinical and Laboratory Standards Institute. Criteria for laboratory testing and diagnosis of human immunodeficiency virus infection; approved guideline, vol. 30 (21). 2. Association of Public Health Laboratories. Public health laboratory issues in brief: 2009 diagnostic survey; 2010. 3. Devaux I, Alix J, Likatavicius G, Herida M, Nielsen S, Hamers FF, et al. Human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) case reporting in the World Health Organization European
10.
11. 12. 13.
14.
Region in 2006. Euro Surveill 2008;13(39), pii 18988. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=18988. Mortimer PP, Parry JV, Mortimer JY. Which anti-HTLV III? LAV assays for screening and confirmatory testing? Lancet 1985;2:873–7. McElborough D. Importance of using an HIV Ag/Ab combined assay in a UK population at high risk of acquiring HIV infection. Commun Dis Public Health 2004;7(4):312–4. Perry KR, Ramskill S, Eglin RP, Barbara JA, Parry JV. Improvement in the performance of HIV screening kits. Transfus Med 2008;18:228–40. Pilcher CD, McPherson JT, Leone PA, Smurzynski M, Owen-O’Dowd J, PeaceBrewer AL, et al. Real-time, universal screening for acute HIV infection in a routine HIV counselling and testing population. JAMA 2002;288(2): 216–21. British HIV Association (BHIVA). 2005–6 mortality audit. www.bhiva.org. Health Protection Agency (HPA), Centre for Infections, The UK Collaborative Group for HIV and STI Surveillance. Testing times. HIV and other sexually transmitted infections in the United Kingdom; 2007, http://www.hpa. org.uk/web/HPAweb&HPAwebStandard/HPAweb C/1203084355941. Sanders GD, Bayoumi AM, Sundaram V, Bilir SP, Neukermans CP, Rydzak CE, et al. Cost-effectiveness of screening for HIV in the era highly active antiretroviral therapy. New Engl J Med 2005;352:570–85. British HIV Association (BHIVA). UK national guidelines for HIV testing 2008; 2008, www.bhiva.org. Sam Moses.Oral presentation—Clinical Virology Network. 2011. Terrence Higgins Trust. Community HIV testing: the feasibility and acceptability of assertive outreach and community testing to reduce the late diagnosis of HIV. Available from: www.bhiva.org/documents/conferences/bournemouth2011/ presentations/110407/O9MichaelBrady.pdf. Health Protection Agency. Anti-HIV screening—minimum testing algorithm. National Standard Method VSOP 11 Issue 2; 2009, http://www.hpastandardmethods.org.uk/pdf sops.asp.
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
HIV testing—The perspective from across the pond夽 Gary Murphy a,∗ , Celia Aitken b a b
Microbiology Services, Health Protection Agency, 61 Colindale Avenue, Colindale, London NW9 5HT, UK West of Scotland Specialist Virology Centre, Gartnavel General Hospital, 1053 Great Western Road, Glasgow G12 0YN, UK
a r t i c l e Keywords: HIV Testing algorithms
i n f o
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 has been a dramatic increase in the types and numbers of assays available for HIV testing coupled with improvements in the sensitivity and specificity of these assays. However, with this increase in choice the algorithms for the initial diagnosis and confirmation of HIV infection have also increased in diversity and complexity and no uniform algorithm exists. Different regulatory regimes have meant that different assays and assay formats are available worldwide. In the UK we have been fortunate in having access to the so called 4th generation HIV antigen/antibody assays for 10 years. The first 4th generation assay in the US was licensed last year. The availability of this class of assays has led to the development of new algorithms for use in the US market and this paper describes how after many years of diversity the HIV algorithms between the UK and US are now converging. © 2011 Published by Elsevier B.V.
The publication of the CLSI (Clinical and Laboratory Standards Institute) document M53-A: Criteria for Laboratory Testing and Diagnosis of Human Immunodeficiency Virus Infection: Approved Guideline provides extensive up to date guidance on laboratory methods and their interpretation in HIV infection.1 The evidence for the recommendations has been gathered over the last five or so years by both the Association of Public Health Laboratories (APHL) and the Centers for Disease Control and Prevention (CDC, Atlanta, GA).2 Regulatory constraints e.g. delays in approval of the 4th generation diagnostic assays have led to a greater divergence between the laboratory procedures in the United Kingdom (UK) and United States (US), and while there have been revisions to the testing guidelines the diagnostic algorithm in the US has remained largely unchanged since 1989. Following FDA approval for the 4th generation assays, it is likely that over the next few years there will be a greater convergence of the 2 countries’ approaches to HIV diagnosis. Even within Europe there are different approaches to testing and confirmation as shown by Devaux et al.3 (Table 1). Local guidelines/algorithms have largely been adapted as new developments have occurred with many institutions implementing changes ahead of any local/national guidance. Although in the US guidelines are generally set by the CDC in collaboration with other organizations, in the UK it is not always clear where the official
夽 Except where cited the views are those of the authors. ∗ Corresponding author. E-mail addresses: [email protected] (G. Murphy), [email protected] (C. Aitken). 1386-6532/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.jcv.2011.09.027
recommendation should come from. Several organizations including the Department of Health, Health Protection Agency (HPA), and HIV organizations such as The British HIV Association (BHIVA), all produce guidance. HIV services in the UK are provided both in teaching hospitals and district general hospitals. The laboratory work supporting these services are performed either in a dedicated virology or microbiology laboratory. Most dedicated virology services are based in teaching hospitals, and in addition to providing virology services for their own patients they will also provide a reference service for local microbiology laboratories. The HPA is an independent UK organization established in 2003, whose primary remit is to “protect the public against health hazards and infectious diseases”, in Scotland this is devolved to HPS (Health Protection Scotland). HPA Colindale is responsible for specialist infectious diseases surveillance, including blood borne viruses, it advises at both the local and national level and is responsible for the writing and dissemination of the National Standard Methods and Algorithms. These algorithms are regularly reviewed and updated as required. They offer laboratories across the country a minimum standard to follow, however they are not mandatory. This article will focus on HIV testing from the UK perspective; it will not deal with screening for organ or blood donors. 1. Historical perspective Ever since 1985, HIV screening assays have relied principally on the detection of infection through demonstrating the presence
S72
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Table 1 HIV testing algorithms used in the countries in the WHO European Region, 2006. Screening test
Confirmation test
No. of countries
ELISA
No test 2nd ELISA Western blot Immunoblot Other 2nd + 3rd ELISA or other
2 17 34 13 5 4
PCR p24 antigen Viral culture
10
Source: Devaux I, Alix J, Likatavicius G, Herida M, Nielsen S, Hamers FF, et al. Human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) case reporting in the World Health Organization European Region in 2006. Euro Surveill 2008;13(39), pii 18988. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=18988.
of anti-HIV antibodies in the serum of the subject under test. The early assays (1st and 2nd generation) detected IgG only with the main difference being the source of the viral antigen—1st generation assays relied on crude or purified viral lysate from HIV-1 only, whereas the 2nd generation assays used either synthetic oligopeptides or carefully selected antigens produced by DNA technology to produce selected viral antigens from both HIV-1 and HIV-2. The 1st generation assays lacked sensitivity and specificity4 and were not able to detect the antibody response to the different HIV-1 clades. Further refinements led to the 3rd generation assays which offered the additional advantage of detecting HIV-1/2 IgM and IgG. On average third generation assays were able to detect HIV-1/2 antibodies 9–15 days after the first detection of HIV p24 antigen (Fig. 1). In the UK the introduction of fourth generation HIV-1/2
assays in the National Blood Service and most hospital laboratories has reduced this period even further. This is achieved by the simultaneous detection of p24 antigen and HIV-1/2 antibodies in a single ELISA.5 The superiority of these assays has been recently reviewed by Perry et al.6 and is shown in Fig. 1. In the United States the first 4th generation assay was licensed for use in 2010. This was a major step forward because previously some US laboratories still relied on the relatively insensitive 2nd generation anti-HIV assays while others had switched to the limited number of 3rd generation HIV antibody assays available. The recommendation of the original CDC/APHL HIV testing algorithm required all anti-HIV repeatedly reactive samples to be confirmed by Western blot. This algorithm became outdated once the sensitivity and specificity of the primary screening test surpassed those of the confirmatory assay. The limited but growing access to the newer generation of assays became a driver of change for US testing algorithms. However, to counter the insensitivity of their antibody screening programmes there was some, off label, use of RNA testing of anti-HIV negative specimens in certain populations such as STI clinic attendees where acute HIV infection was likely. To make this process cost effective specimens were pooled for testing. The presence of high viral load present in very early infection means that specimens can be diluted many times, typically 40–90 specimens per pool, without greatly affecting the sensitivity of the assay.7 In the UK because of the presence of 4th generation assays and a standalone p24 antigen assay the use of pooled RNA testing has not been recommended in the diagnostic environment due to the increased risk of false positive results when compared to the only marginal gain in sensitivity. In the US routine viral load testing is not approved for primary screening and diagnosis although one assay, the Gen-Probe Aptima RNA qualitative assay, is licensed for the confirmation of a reactive EIA. The use of
Fig. 1. Improvement in sensitivity of HIV assays. Reproduced from: Perry KR, Ramskill S, Eglin RP, Barbara JA, Parry JV. Improvement in the performance of HIV screening kits. Transfus Med 2008;18:228–40.
G. Murphy, C. Aitken / Journal of Clinical Virology 52S (2011) S71–S76
pooled RNA testing has, however, been implemented nationally in the UK since 2007 for the screening of blood donations and is also used in the US.
Table 2 Factors affecting choice of HIV algorithm. Factor
Reason
HIV prevalence, HIV incidence, prevailing subtype, etc.
In a high prevalence, high incidence area you may want to use an assay that is targeted to identify acute infections. If your population has a diverse subtype present you would want to choose an algorithm that detects this subtype A low throughput laboratory may not be able to justify the cost of automated platforms and thus have to choose different assays Is the appropriate assay licensed for use in a particular country/state? Do local purchasing contracts limit you to assays from one supplier? Is the patient likely to be lost to follow up if a rapid result not obtained, will a patient be able to access care based on the outcome of their test result? Do the staff have the skill and experience to run a full confirmatory HIV service or will they refer reactives to a secondary centre? Is the algorithm to be used for diagnostic testing or population surveillance? What specimen type will be available—serum/plasma, dried blood spot, saliva? Is it cost efficient to perform testing, and if so to what level, or should specimen be referred elsewhere?
2. Factors influencing the need to change algorithms Using the definition of an algorithm as “a step by step problem solving procedure using a defined number of steps” it is clear that no single algorithm can cover the requirements of screening/testing and confirmation for HIV in all settings and patient groups. Consideration also needs to be given of how, if possible, to incorporate the newer assays (e.g. 4th generation assays and nucleic acid amplification tests [NAT] testing) into current algorithms or to the development of new algorithms. The improvement in sensitivity of the newer screening assays means that previously accepted confirmatory tests, such as Western blot, may now fail to confirm specimens from individuals who are truly infected due to their relative insensitivity to the screening assay. Similarly there are now a number of assays using alternatives to blood samples (e.g. oral fluid and dried blood spots) which also need to be included in the diagnostic algorithms. Hence as the sensitivity and range of assays have increased, the complexity of the confirmatory algorithms has increased rather than decreased and has also contributed to the differences in HIV diagnostic algorithms seen worldwide. In addition to the changes seen in technology there have also been significant changes in the clinical management of HIV disease, coupled with a growing awareness that a number of HIV cases were being missed. A recent audit in 2006, by the British HIV Association (BHIVA) showed that 24% of deaths were a direct result of a late diagnosis (CD4 < 200).8 These late diagnoses were in part due to the missed opportunities to test individuals when they visited their family doctor for other reasons but also reflected a lack of awareness, particularly in sexually active gay men, around the need for regular testing. This was supported by national surveillance data confirming that around one third of individuals with HIV were unaware of their diagnosis,9 and in the US it was shown that 50% of transmissions were from people unaware of their infection.10 As a result in the UK it became a political as well as a clinical imperative to “normalize” HIV testing and offer it in a variety of clinical settings with guidance being issued by the Chief Medical Officers of both England and Scotland. BHIVA produced “Guidelines for HIV testing 2008”.11 These guidelines included the following recommendations on whom and how to test:
1. Who to test All patients with a recognisable risk factor e.g. sexual exposure (either lifestyle, or contact with person from country of high prevalence), intravenous drug use, or presence of a clinical indicator disease. Screening of all patients attending antenatal clinic, termination clinics, sexual health clinics, people from high prevalence countries, and people registering with a general practitioner or acute medical admissions where the prevalence is >2 per 1000. Other groups include those who have symptoms that may be related to primary HIV infection and neonatal samples from babies born to HIV infected mothers. 2. How to test The 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 should have two 4th generation assays and at least one assay distinguishing between HIV-1 and HIV.
S73
Testing volumes, automated platforms
Local/national regulations access to assays Local contracts Ease of access to patient follow up/access to care
Staff skills, experience and knowledge
Algorithm purpose, specimen types
Cost
3. Factors affecting choice of algorithm There are many factors (laboratory and clinical) affecting the choice both of assays (screening and confirmation) and how they are incorporated into an algorithm (Table 2). In the UK, population size, local prevalence and workload are important considerations in determining type of screening assay (manual or automated system) and whether confirmation will be offered locally or not. Most laboratories will also need to consider the performance of tests both for general screening and urgent testing as most services will offer screening of antenatal attenders as part of national policy and testing of patients considered to be at risk of infection such as sexual health clinic attenders. Currently available automated platforms and assay formats allow same day testing to be performed where the laboratory is close to the clinic and an urgent HIV result can often be obtained in 2–4 h. Where such platforms are not available, where specialized laboratory facilities are not available or when a particularly urgent screening result is required, such as during labour when the antenatal screen result is missing or in STI clinics, there has been an increase in the uptake of point of care and rapid HIV testing although a laboratory confirmation of infection is still required. Laboratory services in the UK are currently under review, and this is likely to affect both the size and skill mix of most laboratories. Whereas virology centres will have the expertise to perform screening for HIV and confirmation, this may not be the case for all microbiology laboratories, in these cases they will need to be able to refer their samples onto specialist centres. This will undoubtedly impact 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
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Fig. 2. UK national HIV screening algorithm. Source: Health Protection Agency. Anti-HIV screening—minimum testing algorithm. National Standard Method VSOP 11 Issue 2; 2009, http://www.hpastandardmethods.org.uk/pdf sops.asp.
the individual laboratory.14 An example of a possible confirmatory algorithm is shown in Fig. 3, it is important that the local clinical team understand at what stage a sample is classified as confirmed. Within the UK the accepted screening test is a 4th generation assay on blood. No nationally approved algorithm exists for screening
with other specimen matrices or with rapid tests. This is in contrast to the algorithms produced by CLSI which describe 6 different algorithms describing the type of assay used for the initial screen and covering different sample types such as oral fluid.
Fig. 3. Example of a possible dual 4th generation HIV screening/confirmatory algorithm.
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Fig. 4. Proposed unified US HIV testing algorithm.
In the UK, on identification of a reactive specimen, repeat testing of this sample with either the same test or one of equivalent sensitivity is recommended. At this point full confirmation with an alternate assay should then be performed either in the same laboratory or referred elsewhere (Fig. 3). As discrepant results may indicate very early infection, the algorithms direct the laboratory either to requesting a further sample 1–2 weeks later or NAT detection. The uptake of 4th generation assays in the United States has led to a proposed new testing algorithm (Fig. 4) which now shows greater concordance with the algorithms currently in use in the UK for laboratory confirmation. A recent audit (2010) of diagnostic tests in the UK revealed that the bulk of HIV testing is being carried out in non specialist virology centres, i.e. microbiology facilities (only 35 of 122 labs surveyed were designated Clinical Virology Centres, the remainder were virology units or microbiology laboratories). Nearly all the laboratories surveyed (90%) test antenatal samples in addition to samples from other patient groups. 87% of laboratories test >5000 samples/year with 20% doing more than 20,000 tests/year. Despite guidance, 3 out of the 122 laboratories surveyed still use a 3rd generation assay for screening. Of those laboratories performing confirmation (n = 51) only 33 distinguished between HIV 1 and 2 (the remainder sent samples for testing elsewhere) and in 5 of them an insufficient number or combination of confirmatory assays were used.12 The Western blot as part of a confirmatory algorithm is only used in the 2 Scottish reference laboratories, where it is used as an extra test in addition to the 3 assays used for confirmation. 5. Other considerations In the UK confirmation of patient identity is also critical; this is achieved by testing the original sample from the clot and requesting a second sample. Further samples are also requested when the serological profile is unusual, or when acute infection is suspected. In the latter situation most laboratories would perform NAT testing themselves (either locally or refer the sample to a specialist centre). However it should be remembered these commercial viral load assays are not licensed for this use, although, one of these assays does have a licence for use as a confirmatory test in the US. False positives with very low viral loads have occasionally been reported in this situation.
As stated there are no national algorithms in the UK for the use of rapid/near patient testing or for alternative specimen types to blood. However, rapid/near patient testing is being piloted across a number of different health care settings, and are used in some confirmatory algorithms within local laboratories. Preliminary data support the use of these devices providing there is the appropriate support for patients and the necessary quality control.13 Currently their use in non hospital settings is increasing but there is not any data on how widespread this is. Within laboratories the audit mentioned above12 has shown that 24 out of 133 laboratories use rapid testing devices, with 9 laboratories using 3rd generation assays (6 of which have incorporated it into their confirmatory algorithm) and 15 laboratories use a 4th generation assay (5 of which have incorporated the 4th generation RTD as part of their laboratories’ standard confirmatory algorithm rather than just as a screening assay). In addition to HIV diagnosis in England and Wales the Health Protection Agency has introduced a programme to test every new HIV diagnosis for evidence of recent HIV infection. A similar program is starting in Scotland. This program uses avidity testing to distinguish between recent (last 3 months) and past infection, and is the only program that also returns the result to the patient (Scotland has still to decide whether to anonymize the program or not). The data can be compared to the New HIV Diagnosis database and will allow the estimation of HIV incidence in different populations. Identifying patients with recent infection also allows for targeted contact tracing thereby identifying those individuals at greatest risk when the index was at their most infectious. Other countries such as France and the United States also have programmes for identifying recent infection but these use different laboratory techniques and anonymized specimens. 6. Conclusions Since the discovery of HIV there have been tremendous advances in the care of HIV infected patients. Despite these advances it has been difficult to have common algorithms for diagnosis 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 complicated the establishment of testing algorithms. Following FDA approval of 4th generation assays in 2010, it is likely that there will be fewer
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differences between the algorithms in the US and UK, with most differences being in the detail, reflecting local clinical need.
4.
Funding
5.
None.
6.
Competing interests No financial disclosures were reported by the authors of this paper.
7.
8. 9.
Ethical approval Not required. References 1. Clinical and Laboratory Standards Institute. Criteria for laboratory testing and diagnosis of human immunodeficiency virus infection; approved guideline, vol. 30 (21). 2. Association of Public Health Laboratories. Public health laboratory issues in brief: 2009 diagnostic survey; 2010. 3. Devaux I, Alix J, Likatavicius G, Herida M, Nielsen S, Hamers FF, et al. Human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) case reporting in the World Health Organization European
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