Two weeks ago aidsmap reported that the World Health Organization was recommending a major change towards more frequent use of RNA (i.e. viral load) testing for people with HIV in lower-income settings. This change is necessary, because relying on symptoms or CD4 counts alone to detect treatment failure results in people spending too much time on failing regimens and developing drug resistance. And it is now possible, because RNA test technology is finally becoming cheap enough to use in the field.
Other speakers at the 12th International AIDS Society Conference on HIV Science (IAS 2023), held in Brisbane in July, also urged the use of RNA test technology in PrEP users and especially those on injectable PrEP. This is primarily due to continuing concerns about the occurrence of drug resistance in people who acquire HIV infections despite taking PrEP, often because such HIV ‘breakthrough’ infections take a long time to detect.
But it also addresses more general dilemmas in PrEP – such as whether, for someone who discloses several recent high-risk exposures to HIV, to offer PrEP, offer them a course of post-exposure prophylaxis (PEP) first, or tell them to come back in a week or two, by which time antibody tests will hopefully detect HIV if it is there?
Although there is still no test that can tell if someone is in the first 10-12 days of an HIV infection, RNA tests can minimise the ‘window period’. Also, because they detect unmistakable viral genetic activity, they may be able to offer a confirmed diagnosis in situations where antibodies lack sensitivity, are slower than usual to appear or offer contradictory results, and antigen tests lack specificity.
How many people who acquire HIV on PrEP develop resistance?
Dr Urvi Parikh of the University of Pittsburgh is PEPFAR’s lead on drug resistance. She told the conference that “the fear of resistance should not drive us to restrict access to PrEP”. Nonetheless, she added, there were concerning signs that introducing injectable cabotegravir – an integrase inhibitor – into PrEP programmes might lead to an increase in what has up until now been an uncommon form of resistance.
In the scientific studies and demonstration projects of oral PrEP using the nucleoside reverse transcriptase inhibitor (NRTI) drugs tenofovir disoproxil and emtricitabine, between 4 and 7% of participants who acquired HIV despite accessing PrEP developed drug resistance – most frequently the M184V/I resistance mutation to emtricitabine and/or sometimes K65R or similar mutations against tenofovir. But in full PrEP rollouts, the proportion of people developing resistance has risen to 22% – mainly because people are being less intensively tested. (The prevalence of drug resistance among people who turned out to have just caught HIV before they started PrEP is much higher – between 40 and 65%.)
However, these resistance mutations do not usually have serious clinical consequences. The K65R mutation only reduces susceptibility to tenofovir by 50%, and while HIV with the M184V mutation is completely resistant to emtricitabine, it actually increases its susceptibility to tenofovir. In any case, it is not difficult to construct an antiretroviral therapy (ART) regimen that does not use drugs from the NRTI family.
But integrase inhibitors such as dolutegravir, bictegravir and cabotegravir are now the cornerstone of ART in both high-income and low-income settings.
Resistance to integrase inhibitors has hitherto been much rarer than to NRTIs. In a US survey the prevalence of integrase mutations in transmitted viruses was only 0.2%, and none were found in a UK survey – compared with an 8% prevalence of NRTI mutations.
But in an Italian survey of patients for whom integrase-inhibitor based treatment had failed and who had acquired resistance, 15% had treatment-limiting integrase resistance, defined as loss of susceptibility to dolutegravir, which is more resistance-proof than cabotegravir.
Urvi Parikh presented an update on infections in the open-label extension of the pivotal HPTN 083 study of cabotegravir PrEP in gay and bisexual men and trans women. There have now been 110 infections in the 4266 participants (76 in those taking TDF/FTC pills and 34 in people receiving cabotegravir injections).
Among the 14 participants whose last cabotegravir injection was more than six months prior to infection, there has been no integrase inhibitor resistance and only one case of delayed HIV seroconversion (the appearance of testable antibodies).
But among the 18 people infected less than six months after their last injection, there have been 14 cases of delayed HIV seroconversion and (out of 16 tested for it) ten cases of clinically relevant integrase inhibitor resistance.
Resistance was acquired by all six people with 'breakthrough' infections, in other words those who became infected despite having on-time injections and satisfactory cabotegravir levels. (Although the transmission of a virus that was already cabotegravir resistant can’t be ruled out, such infections are very rare.) Four people had only one mutation, which meant dolutegravir might still work, but the other six acquired multiple mutations, effectively ruling out integrase inhibitor-based ART.
As already mentioned by HPTN 083 researchers at CROI 2022, using RNA testing for people on injectable PrEP could have diagnosed their HIV infections a lot earlier and resistance might possibly have been avoided. Instead, RNA testing was only used to retrospectively test samples from people who tested positive using a conventional rapid antibody test.
The average gap between the first sample containing detectable HIV RNA and a positive rapid test in people with delayed positivity was 75 days, with a range from 28 to 185 days – in other words, between one and six PrEP injections were received while the person already had HIV.
Needed: updated testing algorithms for PrEP
Dr Jason Reed of the international non-profit health organisation Jhpiego told the conference that testing protocols needed to be updated for injectable PrEP. At present there was ‘discordance’ between what the US Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC) recommended, and the testing guidelines used by other countries, generally based on WHO’s algorithm.
The CDC guidelines on injectable PrEP say: “Because of the long duration of drug exposure [on injectable PrEP]…exclusion of acute HIV infection is necessary with the most sensitive test available, an HIV-1 RNA assay”. The FDA says a test “cleared by the FDA for the diagnosis of acute HIV infection” should be used. This could be an RNA test, but if a fourth-generation test which detects p24 antigen and antibodies is used, the result will need to be confirmed with RNA test. If the person has been on oral PrEP in the last three months or injectable PrEP in the last 12, the CDC recommends that both a fourth-generation test and an RNA test are performed.
In contrast, WHO only recommends two rapid antibody tests, one with high sensitivity to exclude false negatives and the second with high specificity to exclude false positives. If the first test is negative, that’s the end of the story. If it’s positive and so is the second, a sample is usually sent to a central facility for an RNA or other confirmatory test. If the first two tests have divergent results, the first test is repeated and if they are still divergent, the advice is to wait 14 days to see if they both become positive. While this algorithm is suited to a normal course of events where discordant test results are usually due to very recent infection and should soon harmonise, with the main concern being to avoid false positive results, it is clearly less suitable for a situation where people may test false negative for a prolonged period.
Jason Reed said that there were now a sufficiently large number of people in injectable PrEP rollouts and in new scientific studies and demonstration projects to address our ignorance of the best testing methods to prevent delayed detection.
Eleven countries so far will receive at least some support from PEPFAR for injectable PrEP before 2025. They will generally use the WHO testing algorithm: they are mainly in Africa but also include Ukraine, Thailand and the Philippines. But there are also 11 scientific studies planned or underway that will look not only at injectable PrEP but at combinations of oral, topical and injectable PrEP. They have the opportunity to use RNA testing too. An example is the CATALYST study which will compare the efficacy of oral and injectable PrEP and the dapivirine vaginal ring in women in Kenya, Lesotho, South Africa, Uganda, and Zimbabwe, and there are also studies planned among gay and bisexual men and trans women in Vietnam and Brazil.
All but one of the 11 studies will use a conventional third-generation rapid antibody test, but five will also use the fourth-generation antibody/antigen test, which cuts down the ‘window period’ from about three weeks to two by detecting the p24 viral protein, and eight studies will include RNA testing. Four will use both fourth-generation and RNA tests.
What HIV RNA assays can, and cannot detect
Dr Lucia Hans of the University of the Witwatersrand in South Africa looked more deeply into the HIV RNA assays that are often used in lower-income settings and whether they would be sensitive enough to pick up early HIV infection where viral load is being suppressed by PrEP.
At the time of their RNA test, the people who caught HIV on cabotegravir had very different viral loads. Among the four people who already had acute HIV when they started PrEP, the lowest viral load was 1400 (average 24,900) and among those who acquired HIV due to delayed injections the lowest viral load was 48,000. The two who acquired HIV just before resuming injections had viral loads of 450 and 1620 so should also be detected by a conventional plasma RNA test – even, possibly, by a dried blood spot assay.
But the mean viral load of the three people who caught HIV while on the initial period of oral cabotegravir was 210 and of the six people who had breakthrough infections it was 390. However, the lowest viral loads were 16.3 and 6.1 – only quantifiable by an ultrasensitive assay. There was also one viral load listed as “below 40”, in other words detected but unquantifiable by the test used.
What in fact are the lower limits of detection and of quantification for the most widely used RNA assays, especially where only alternatives to plasma samples (which need to be centrifuged and refrigerated) are available? Lucia Hans listed four qualitative assays in widespread use. Qualitative assays are ones that give a simple "yes, detected" or "no, not detected" answer. The lower limits of detection using dried blood spots were 255, 531 and 2500 respectively on three different qualitative assays, and 278 and 2491 on two assays using whole blood.
Quantitative assays give a precise figure for viral load. In the case of four assays cited by Hans, the manufacturers’ reported lower limits of quantification, using dried blood spots, ranged from 400 to 873. (Note that this lower limit of quantification is lower than the 1000 copies/ml suggested by WHO as the threshold for a “suppressed viral load” and in two cases would be sensitive down to viral loads that have never been found to lead to transmission.) All these have much lower limits of quantification if plasma samples are used, from 20 to 50, though one Hans listed has a lower limit of detection of 13.5 copies, though it can’t quantify a viral load below 40.
With dried blood spot samples, therefore, these assays would have detected all the infections in cabotegravir recipients in HPTN 083 who had infection at baseline or who had delayed injections, but none of the oral-phase infections and only one of the breakthroughs. With plasma samples, they would have detected two more of the breakthrough infections and two of the oral-phase infections; but two and possibly three participants’ infections were only detected with laboratory-based hypersensitive assays that need larger samples and considerable expertise to run.
Viral load testing facilities are now available in most lower-income countries and there are only 13 countries where it is known (data is missing for some others) that viral load testing is available at less than 50% of HIV clinical sites. These include eight countries in Africa, the biggest being the Democratic Republic of Congo, but also include Honduras and Belize in central America and Iraq and Bangladesh in Asia. Only two test manufacturers gave figures for availability in Africa; Abbott said they had 90 active assay systems in Africa, though a third were in South Africa, and Roche had 125, of four different types. The Maghreb and Saharan countries appear to be the least well served.
Conclusions: HIV tests and the 'new PrEP'
Over the next few years we should see the start of RNA testing in PrEP programmes, especially programmes offering long-acting injectable PrEP, as well as in HIV treatment, though it may take longer to implement.
We will probably not see viral load tests being used for every routine three-monthly test in PrEP; we will still depend on PrEP users’ accounts of whether they may have had an infection risk, or are struggling to attend appointments/take the pills, in conjunction with antibody testing.
However, Lucia Hans had one word of warning. At present the orthodoxy in countries like South Africa is to integrate PrEP and HIV treatment services, both for reasons of economy and to avoid stigma. However, at present the small number of PrEP-related viral load tests being done is overwhelmed by the hundreds of thousands of viral load tests being done for people on ART. She felt that the future of PrEP should ideally be a separate HIV prevention and testing service.
During questions and answers, the speakers conceded that the existence of LEVI syndrome means that we may never be able to avoid all situations where someone who already has HIV takes PrEP, and there was still no substitute for incorporating patients’ accounts of exposures to HIV and close attention to symptoms suggestive of acute infection into decisions such as whether to start PrEP or suggest PEP; whether to stop PrEP and start ART in the presence of indeterminate results; and other dilemmas. In the longer-term future we may have ‘TNA’ assays that can measure viral DNA as well as RNA, and these may get as close as we can come to finding out if someone really does have HIV.
Parikh U. Long-acting PrEP and the potential for resistance
Reed J. Long-acting PrEP: Programmatic considerations for HIV testing
Hans L. HIV testing approaches: Current and new approaches and considerations for long-acting PrEP.
All presentations in HIV testing in the context of long-acting extended delivery of HIV PrEP, Symposium Session number SY11, 12th IAS Conference on HIV Science, Brisbane, 2023.