atypical hiv test results when prep is prevalent – a need for vigilance in the laboratory

issue: hiv and aids

06 november 2018 article

mt-nov-18-murphy,-brown,-bradshaw,-saunders,-gill.jpg

since the first diagnostic test for hiv was licensed in 1985, sensitivity and specificity has improved enormously, shortening the time taken to identify someone as having acquired hiv.

typically, hiv infection leads to a high-titre viraemia within two to three weeks of infection, and within a few days of the virus becoming detectable, a protein component of the virus core, p24ag, is usually detectable. the presence of p24ag stimulates the humoral immune response to begin developing igg and igm responses. the igm response falls after one to two weeks, but igg increases in intensity over many months, until years later there is degradation of the overall immune response associated with progressive hiv-related disease. using ‘fiebig’ staging, the maturation of the hiv-specific immune response following initial infection can be used to categorise individuals as acutely infected, or having a longer-term antibody response.

antiretroviral therapy and hiv antibody response

very early initiation of effective antiretroviral therapy (art) may inhibit the performance of diagnostic assays during the initial course of infection. as the maturation and maintenance of an antibody response is strengthened by sustained antigenic stimulation, such early use of art can inhibit this response by very quickly reducing the amount of virus. this leads to less antibody production, and less time for antibodies to mature. therefore, the diagnostic test results seen in these infections may be ‘atypical’. responses may happen later, not at all, or at levels lower and more fragmented than usually seen. reactivity on two or more consecutive samples may not change or may stay discrepant while remaining on prep or after stopping. in rare cases, early treatment can lead to sero-reversion where, although still infected, there is no detectable antibody response on serum testing. these diagnostic issues need consideration when testing specimens from those exposed to, or confirmed as acquiring, hiv infection while also receiving art as either post-exposure prophylaxis (pep) or very early treatment initiation.

pre-exposure prophylaxis and scale-up

pre-exposure prophylaxis (prep), which currently involves individuals taking a tenofovir preparation combined with emtricitabine, has been shown to be highly effective at protecting against acquiring hiv-1 infection. infections that do occur will usually happen when prep has been used inconsistently, very rarely during consistent use, or may appear to happen if infection is acquired shortly before prep beginning but is recognised later. in each case the stimulus to the immune response may be altered, producing an atypical antibody response.

opportunities for accessing prep have multiplied across the uk since 2015, leading to an accelerating scale-up of the numbers of gay men using the intervention. although data on atypical results has not been centrally collated, it is probable that there has been concomitant increase in the likelihood of atypical hiv test results. prep is available on private prescription, can be bought directly at low price from one london clinic or from overseas, and publically funded prep programmes have begun in scotland, wales and northern ireland. in england, the prep impact trial, a pragmatic health technology assessment, is addressing outstanding questions on prep eligibility, uptake and duration of use, and impact on hiv and other stis. since beginning in mid-october 2017 and june 2018, almost 7,900 participants, mostly gay men, were enrolled at 140 clinics, and another 5,100 trial places are available. while 9,854 pep courses following sexual exposure were begun by gay men in england in 2017, it is probable that over 10,000 gay men are now taking prep regularly. there is increasing evidence from a variety of groups, particularly those who test large volumes of blood donors, and from recent data from prep trials that pep, prep and art initiation during acute infection can cause blunting of the hiv-1 antibody response. both non-reactive hiv serology and non-progressive fiebig profiles have been seen, in a situation where detectable virus in the blood is also unlikely.

the diagnostic challenge for those taking prep with atypical test results

diagnosing ‘breakthrough’ hiv infections, therefore, may be a complex task. in analysing a sample from someone taking prep, it will be critical to consider all atypical results (box 1) as indicating potential infection and to manage this accordingly. it will take time and multiple tests, including western blot antibody analysis, as well as rna and proviral dna molecular assays to determine whether these patients with ‘atypical’ or discrepant antibody profiles have acquired hiv. it is too soon and the data are too sparse for reaching a consensus on which diagnostic assays, if any, will perform better in the era of prep. the number of reported breakthrough infections while on prep (four by march 2018), and incomplete recording to date of those with any form of ‘indeterminate’ antibody result while taking prep, means very few platforms and assays have been assessed. this july at aids 2018, a session was held to highlight the importance of the topic and emphasise the work that needs to be done.

the decision about whether an individual with an atypical hiv test result should remain on prep or receive additional art to treat a potential infection is important for individual management, but may compromise the opportunity to confirm a diagnosis. atypical results may also contribute to a patient with hiv-1 beginning a potentially less efficacious treatment regimen. it is also important to consider the interpretation of the hiv-negative results that these individuals receive. is the result truly negative, or could it be a consequence of the blunting of the immune response? any sudden increase in the level of reactivity in a repeat sample in a diagnostic assay, even if still below the negative cut-off, should be considered suspicious and monitored. clinicians managing individuals with atypical hiv test results (box 1) while taking prep should be advised of the need for repeat testing with combined antigen/antibody assays, or with molecular methods, on cessation of prep to ensure hiv infection has not occurred. these atypical testing cases should be discussed with a regional expert and investigated further for possible seroconversion. follow-up testing should take account of the ‘high-risk’ testing window, i.e. retest at both 4 and 8 weeks following discontinuation of prep.

the clinical services unit of public health england (phe) colindale would welcome information of such cases (via an email without personal identifying information sent to [email protected]). if requested, the unit will liaise with the regional expert, provide expert advice, and collate information on the frequency and details of these events. phe, in collaboration with colleagues at imperial college, already investigate individuals who demonstrate unusual immune responses to hiv (box 2). this service for managing individuals taking prep who have atypical test results will continue as prep scale-up proceeds further, testing specimens on a variety of diagnostic assays. these tests will cover a range of hiv-specific antigens to help determine if there is preferential reactivity against any individual antigen. such evidence may guide the make-up of diagnostic assays that should be used in prep monitoring or screening programmes.

if a seroconversion event is suspected while taking prep, current best practice is to intensify art while continuing laboratory investigations. if an atypical result is first detected after prep has been stopped, then it is advised that no further prep is prescribed until an expert consensus is reached regarding the individual’s hiv status.

laboratory request forms submitted with samples for further virological investigation (including hiv viral load testing or combined antibody/antigen testing) should contain information on whether the patient has been taking either pep or prep, and if so, when and for what duration, to allow for better interpretation of atypical results.

conclusion

the prep era marks a potential paradigm shift in hiv testing, where for a small population of cases persistent or one-off low-level antibody reactivity will require more follow up and consideration. international consensus and experience sharing will be key to highlight the best testing strategies for this group of at-risk individuals, and to ensure they have the best information about what any indeterminate result may mean.

box 1: atypical hiv results: what to look for

1.	low signals near to cut-off in screening assays (including either just below or below cut-off).
2.	sero-reversion on follow-up specimens.
3.	discrepant results between assays.
4.	slow development of antibody/antigen signal in subsequent samples.
4.	weak and/or incomplete banding patterns on innolia or western blot.

box 2: hiv reference laboratory services at phe colindale

1.	wide range of assays (non-standard commercial and in-house elisas, proviral dna, novel sequencing).
2.	western blot to determine antibody-specific responses.
3.	collation of test results from a variety of platforms to determine prep interference with particular assays.
4.	referral to clinic specialising in atypical serological responses to hiv infection (difficult diagnoses).

gary murphy

national infection service laboratories, public health england, 61 colindale avenue, london nw9 5ht, uk

[email protected]
facebook: publichealthengland
twitter: @phe_uk

gary murphy is joint scientific lead of the phe clinical services unit. he has an interest in hiv incidence and laboratory diagnostics which stemmed from watching quincy as a child and wanting to be just like sam, the trusted lab scientist.

colin s. brown

national infection service laboratories, public health england, 61 colindale avenue, london nw9 5ht, uk

[email protected]
facebook: publichealthengland
twitter: @phe_uk

colin s. brown is a consultant microbiologist. colin was inspired to work in the field of hiv given the opportunity to make a difference at individual, population and policy levels. his varied interests mean when not in colindale he can often be found supporting projects in west africa.

daniel bradshaw

national infection service laboratories, public health england, 61 colindale avenue, london nw9 5ht, uk

[email protected]
facebook: publichealthengland
twitter: @phe_uk

daniel bradshaw is a consultant in the phe virus reference department. he trained as a sexual health physician before undertaking a post-cct fellowship in virology. his main interests are hiv and viral hepatitis.

john saunders

public health england sti & hiv department, 61 colindale avenue, london nw9 5eq, uk

[email protected]
facebook: publichealthengland
twitter: @phe_uk

john saunders is a sexual health and hiv clinical academic working at public health england, university college london and the mortimer market centre in central london. he has a wide range of scientific interests and is clinical champion of the national chlamydia screening programme.

noel gill

public health england sti & hiv department, 61 colindale avenue, london nw9 5eq, uk

[email protected]
facebook: publichealthengland
twitter: @phe_uk

noel gill is head of the hiv and sti department at public health england, colindale and has a special interest in hiv. his paper ‘the hazard of infection from the shared communion cup’ gave him the knowledge base and confidence to enter the hiv field in late 1987.

on a typical day in this position, what do you do?

our day jobs include seeing patients, managing a high-throughput diagnostic laboratory, interpretation of lab results, service development and extracting information from carefully constructed tables summarising data. these are all done with the aim of improving hiv diagnosis and reducing the burden of hiv disease – a topic which unites us all.

what do you love most about your job?

working closely together we are able to bring expertise from different disciplines into focused actions that bring benefits on individual and population levels.

image: technician holding a multi-well sample tray as he tests blood for hiv. tek image/science photo library.

back to issue

atypical hiv test results when prep is prevalent – a need for vigilance in the laboratory

antiretroviral therapy and hiv antibody response

pre-exposure prophylaxis and scale-up

the diagnostic challenge for those taking prep with atypical test results

conclusion

further reading

box 1: atypical hiv results: what to look for

box 2: hiv reference laboratory services at phe colindale

gary murphy

colin s. brown

daniel bradshaw

john saunders

noel gill

on a typical day in this position, what do you do?

what do you love most about your job?