Breakthrough Acute HIV Infections among Pre-Exposure Prophylaxis Users with High Adherence: A Narrative Review

Pre-exposure prophylaxis (PrEP) is a pivotal intervention among HIV prevention strategies. We aimed to narratively revise the topic of HIV acute infection in the setting of PrEP exposure with a focus on diagnostic options, clinical features, and future PrEP perspectives, with a particular focus on users with high adherence to PrEP. We searched the main databases (PubMed, Embase, and Scopus) with the keywords “PrEP” or “Pre-Exposure Prophylaxis” and “HIV” or “PLWH” and “breakthrough” or “acute infection” or “primary infection”. We included all randomized clinical trials and non-experimental studies (both case reports and observational studies) ever published. In the present narrative review, we revise the diagnostic challenges related to HIV diagnosis in the setting of PrEP and the clinical characteristics and symptoms of breakthrough infections. We discuss the management of acute HIV infection during PrEP and the new challenges that arise from the use of long-acting drugs for PrEP. Our review underlines that although extremely rare, HIV seroconversions are still possible during PrEP, even in a context of high adherence. Efforts to promptly identify these events must be included in the PrEP follow-up in order to minimize the chance of overlooked HIV breakthrough infections and thus exposure to suboptimal concentrations of antiretrovirals.


Introduction
Forty years since the first reports of AIDS, the therapeutic advancements in the field of HIV have granted people with HIV (PWH) a life expectancy similar to that of people without HIV and an improved quality of life [1].The key factor to successful treatment is an early diagnosis with a prompt start of antiretroviral treatment.This is pivotal for both overall mortality and comorbidity reduction and to reduce the risk of transmission.In particular, an international cornerstone study, the INSIGHT START study, showed how patients who started antiretroviral treatment had a significantly lower risk of developing severe AIDS-related events compared to patients who delayed the start of treatment, regardless of their CD4 cell count [2,3].Despite these advancements, however, new HIV acquisitions are still a public health challenge worldwide.In particular, despite the different preventive strategies that are available nowadays, recent data from UNAIDS show that we are still far from the end of the AIDS pandemic [4].
The use of pre-exposure prophylaxis (PrEP) has quickly arisen as a major player in the fight against the ongoing HIV pandemic.The first pieces of evidence of the effectiveness Viruses 2024, 16, 951 2 of 19 of oral chemoprophylaxis in reducing the risk of HIV transmission date back to over a decade ago, as trials showed high-level protection from the infection while regularly consuming tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) when compared to a placebo [5].As proven by a recent meta-analysis, PrEP has been shown to be particularly effective in men who have sex with men (MSM), with a rate reduction of 75% of new infections; furthermore, the meta-analysis highlighted how adherence was a keystone for treatment success, with the rate of reduction rising up to 86% in trials with high adherence [6].
From the first commercial release of oral PrEP in 2012, its use has steadily increased over the years, becoming widely accessible in most high-income and some lower-income countries [4].While oral tablets are, as of now, the main available tool for chemoprophylaxis against HIV, new options are gaining popularity, such as dapivirine monthly rings, studied for the female population, and cabotegravir-based long-acting PrEP [7,8].
Although PrEP is highly effective, seroconversions have been observed with a variable incidence in trials [9,10].Failure could be a consequence of an unrecognized infection acquired before the initiation of PrEP, low adherence to the oral regimen, or the acquisition of a virus with resistance mutations to PrEP drugs [11,12].
A universal consensus on the definition of "PrEP failure" is still lacking, though it has been described by some authors as "all seroconversions occurring at any time along the care continuum for PrEP" [13].The absence of a clear and consistent definition does not thus fully allow for the comparison of different reports.
We aimed to narratively revise the topic of HIV acute infection (AHI) in the setting of PrEP exposure with a focus on diagnostic options, clinical features, and future PrEP perspectives with a particular focus on users with high adherence to PrEP.

Materials and Methods
We searched the main databases (PubMed, Embase, and Scopus) with the keywords "PrEP" or "pre exposure prophylaxis" and "HIV" or "PLWH" and "breakthrough" or "acute infection" or "primary infection" with the aim of producing a narrative review of PrEP breakthrough infections.We included all randomized clinical trials and non-experimental studies (both case reports and observational studies) ever published.Three reviewers (SL, MLC, and FC) independently screened the titles and abstracts to determine eligibility for full-text review.No geographical restrictions were applied.Only publications in peer-reviewed journals in the English language were included.Studies were included if they met all of the following criteria: (i) study was published in full; (ii) study described PrEP; and (iii) study included any kind of PrEP, such as TDF, TDF/FTC, TAF/FTC, dapivirine ring, and long-acting cabotegravir.Conference papers and abstracts were excluded.Among those, we only selected papers featuring the characteristics of PrEP users with a breakthrough of acute HIV infection with a focus on adherence, diagnostic tools, and symptoms reported.

Diagnostic Tools
HIV early diagnosis has always proven to be a challenge, both from a clinical and a technical point of view.As a variety of new diagnostic tools has been developed over time, each with its own target and sensitivity, it has become crucial for the physician to know which test to use considering the possible timing of infection.
Highly sensitive immunoassays targeting p24 antigen and anti-HIV antibodies represent the standard of care for HIV screening in high-income countries, whereas Western blot (WB) is currently used as the main confirmatory assay [14,15].Aside from serological tests, nucleic acid amplification tests (NAATs) represent, as of now, the gold standard in HIV viral load testing, which is a key prognostic marker for disease progression and the indicator of response to antiretroviral therapy (ART).
In regard to early infection, Fiebig staging provides a descriptive tool based on antigen p24, WB, and HIV-RNA, where each stage describes a unique pattern of assay reactivity [16].Stage 0, or the eclipse period, is characterized by completely undetectable viral markers in blood samples, as it corresponds to the earliest phase of infection, lasting on average 5 to 7 days [17,18].During stage I, HIV-RNA becomes detectable, as all other tests remain negative; during stage II, p24 antigen is also detectable, in addition to NAATs; stage III is characterized by positivity of HIV-RNA, p24 antigen, and IgM-sensitive assays, even though WB is still negative; in stage IV, WB shows an indeterminate pattern, in which the first HIV-specific bands are detectable, though failing to meet the international interpretative criteria of positivity (i.e., at least two of p24, gp41, or gp120/160 are reactive); stage V is characterized by a positive pattern, lacking p31 reactivity; lastly, in stage VI, WB displays a fully reactive pattern, which includes a p31-specific band (this stage indicates an infection acquired within the previous 2 to 3 months) [16,19,20].
Understanding the dynamic of serological and virological assays in the context of PrEP exposure is crucial for an adequate management of people with a suspected AHI while taking PrEP.Indeed, the results of the above-mentioned tests have shown to be influenced by external factors.The PARTNERS study, a retrospective analysis of a controlled, doubleblind, randomized trial of PrEP (TDF/FTC or TDF alone) compared to a placebo, observed a significant increase in the mean time to a positive Western blot in the PrEP group, estimated as 80 vs. 49 days.The same study showed that HIV-RNA viral load was on average 2 or 3 log10 lower in those with AHI in the PrEP group when compared to a placebo [21].These findings suggest that PrEP might delay seroconversion and even hinder HIV-RNA detection during the acute phase of infection, making the diagnosis of AHI a challenge [14].
It is crucial to rule out AHI before PrEP start, as exposure to TDF/FTC in unrecognized HIV infection could make diagnosis more difficult while failing to adequately control viremia.It has also been reported that suboptimal exposure to antiretrovirals carries the potential to select resistance mutations [22].In light of this, most international guidelines emphasize the HIV testing algorithm to be implemented in the context of PrEP initiation.Furthermore, people taking PrEP are supposed to be monitored for HIV with trimestral testing [23].

AHI Clinical Spectrum
AHI can be clinically apparent as a mononucleosis-like illness, which can be referred to as "acute retroviral syndrome".It consists of fever, pharyngitis, generalized lymphadenopathy, weight loss, gastrointestinal manifestations, and a maculopapular, urticarial, or roseolalike rash.Less commonly, a neurological syndrome, such as aseptic meningitis, encephalitis, or peripheral neuropathy with Guillain-Barré syndrome, can be observed.Conversely, opportunistic infections have been seldom described, such as Candida spp.esophagitis or P. jirovecii pneumonia [24].
Symptoms of acute retroviral syndrome usually develop 2 to 4 weeks after HIV exposure (longer timeframes, reaching 10 weeks, have also been described), generally last for 10 to 15 days, and mostly resolve spontaneously [19,24].The clinical manifestation of infection usually precedes peak viremia, typically in a stage in which anti-HIV antibodies have not yet been developed and even p24 is still undetectable in blood.HIV-RNA NAAT is the assay of choice, which allows for detection of the typical high-level viremia observed in this phase.The actual prevalence of symptoms in acutely infected people is still a matter of debate, as most asymptomatic infections remain undetected.The estimated proportion of symptomatic AHI varies widely, typically ranging from 10 to 60% [24,25].
A total of 42 studies were included in the final analysis of this narrative review.Among them, 13 were case reports, 8 were observational studies, and 21 were clinical trials.The full selection process is represented graphically in Figure 1.
A total of 42 studies were included in the final analysis of this narrative review.Among them, 13 were case reports, 8 were observational studies, and 21 were clinical trials.The full selection process is represented graphically in Figure 1.

Breakthrough Acute HIV Infections Reported in Experimental and Non-Experimental Studies
We identified 13 published case reports describing 15 cases of AHI that occurred during PrEP care (Table 1).

Breakthrough Acute HIV Infections Reported in Experimental and Non-Experimental Studies
We identified 13 published case reports describing 15 cases of AHI that occurred during PrEP care (Table 1).Among these cases, 13 individuals were on a TDF/FTC regimen (12 daily, 1 ondemand), while the remaining 2 were on a TDF-alone regimen for HBV-related chronic hepatitis.Most AHIs were identified using a fourth-generation assay with or without HIV-RNA NAAT, with five patients showing clinical signs or symptoms of AHI.Adherence was evaluated using various methods, including therapeutic drug monitoring (TDM), dried blood spot (DBS), hair analysis, pill dispensation records, or self-reporting.By applying a definition of true PrEP failures including any AHI in patients with documented consistent adherence to PrEP, we identified seven cases of true PrEP failure.Of note, three additional cases were consistent with HIV infection acquired prior to the initiation of PrEP in a context of good PrEP adherence.
Regarding the data from observational studies, we identified eight studies that collectively reported 315 cases of seroconversions among a total of 45,947 participants (Table 2).Remarkably, a single study contributed 266 of them, yet it did not assess adherence, leaving 49 seroconversions with available adherence data.Among these, 23 were identified as true PrEP failures.Data regarding clinical manifestations were only available in two of the reviewed studies.

Seroconversions versus True Breakthrough Infections in Clinical Trials
All clinical trials included an adherence assessment.After excluding cases where patients were considered non-adherent to PrEP, a total of 55 AHI were categorized as true PrEP failures.Data regarding clinical manifestations were only available in one of the reviewed studies.Among all of the selected studies, the proportion of seroconversions in oral TDF/XTC PrEP users was 260/30,563 (0.85%), with breakthrough infections in oral TDF/XTC PrEP users (total of cases with adherence available data) accounting for 0.08% (22/28,366).
Focusing on those consuming oral TDF/XTC PrEP with a daily scheme, the overall seroconversions were 254/30,120 (0.84%), with a similar breakthrough infection rate of 22/27,923 (0.08%), while those consuming oral TDF/XTC PrEP with an on-demand scheme had a higher rate of seroconversion (6/443; 1.35%), but no breakthrough infections were reported in this group (Figure 2).
We need to be clear that besides clinical trials, the majority of the studies had a measurement of adherence based on patients' self-reports (Tables 1 and 2).Analyzing topical PrEP (either with tenofovir 1% gel and dapivirine vaginal ring) in the sole context of clinical trials, a total of 42/638 (6.58%) of seroconversions were observed, with 9/638 (1.41%) breakthrough infections.
We need to be clear that besides clinical trials, the majority of the studies had a measurement of adherence based on patients self-reports (Tables 1 and 2).

How to Minimize the Risk of Overlooked HIV Infection during PrEP Start
A crucial step for clinicians before prescribing PrEP is the assessment of HIV status, as, in that moment, the exclusion of unrecognized AHI is of pivotal importance.Recent studies have shown that starting PrEP during an undiagnosed AHI is the main driver of selection for HIV drug resistance, which could potentially complicate subsequent HIV management [66][67][68][69][70][71].
Most international guidelines recommend as a best choice for HIV testing a fourthgeneration antigen/antibody assay conducted by a laboratory and obtained within one to four weeks before initiating PrEP.Repeating the test after one month should also be considered to exclude inadvertent AHI at PrEP start.According to US Public Health Service and BHIVA/BASHH guidelines, a negative blood-based point-of-care (POC) test is acceptable for same-day PrEP initiation, but a laboratory fourth-generation antigen/antibody assay should always be ordered at baseline so that in case of unrecognized AHI the patient can be rapidly transitioned from PrEP to HIV care [72][73][74][75] (Figure 3).

How to Minimize the Risk of Overlooked HIV Infection during PrEP Start
A crucial step for clinicians before prescribing PrEP is the assessment of HIV status, as, in that moment, the exclusion of unrecognized AHI is of pivotal importance.Recent studies have shown that starting PrEP during an undiagnosed AHI is the main driver of selection for HIV drug resistance, which could potentially complicate subsequent HIV management [66][67][68][69][70][71].
Most international guidelines recommend as a best choice for HIV testing a fourthgeneration antigen/antibody assay conducted by a laboratory and obtained within one to four weeks before initiating PrEP.Repeating the test after one month should also be considered to exclude inadvertent AHI at PrEP start.According to US Public Health Service and BHIVA/BASHH guidelines, a negative blood-based point-of-care (POC) test is acceptable for same-day PrEP initiation, but a laboratory fourth-generation antigen/antibody assay should always be ordered at baseline so that in case of unrecognized AHI the patient can be rapidly transitioned from PrEP to HIV care [72][73][74][75] (Figure 3).This timely transition is essential in light of recent evidence suggesting that the M184 mutation can develop more rapidly than thought before, within just 1-2 weeks of TDF/FTC exposure [66].
The risk factors for AHI should be investigated during every initial evaluation for PrEP.Everyone should thus be questioned about engagement in risky behaviors and the presence of signs or symptoms consistent with AHI in the prior 4 weeks.If any of these risk factors are reported, a plasma sample for HIV-RNA NAAT should be sent to increase sensitivity in AHI diagnosis [72][73][74][75].However, it is largely known that during the first 7 to 10 days after HIV infection (the eclipse phase), even HIV-RNA could be undetectable [76].It seems reasonable, then, to defer PrEP start and retest after 2-4 weeks with both a fourthgeneration antigen/antibody assay and HIV-RNA NAAT in case of very recent at-risk exposure or onset of AHI clinical features.Although highly sensitive, HIV-RNA testing is more expensive and not fully free from false positive results, which are likely to be the consequence of a laboratory error [77,78].In such cases, low viral loads are usually detected, and repeating HIV-RNA NAAT on a new plasma sample is recommended by US Public Health Service guidelines in the case of viral loads below 200 copies per milliliter [72].Since the occurrence of false positives cannot be excluded, we also suggest that a low-level viremia on a single plasma sample, without a complementary positive fourth-generation test, should prompt confirmation with an HIV-RNA NAAT on a further plasma sample (Figure 4).This timely transition is essential in light of recent evidence suggesting that the M184 mutation can develop more rapidly than thought before, within just 1-2 weeks of TDF/FTC exposure [66].
The risk factors for AHI should be investigated during every initial evaluation for PrEP.Everyone should thus be questioned about engagement in risky behaviors and the presence of signs or symptoms consistent with AHI in the prior 4 weeks.If any of these risk factors are reported, a plasma sample for HIV-RNA NAAT should be sent to increase sensitivity in AHI diagnosis [72][73][74][75].However, it is largely known that during the first 7 to 10 days after HIV infection (the eclipse phase), even HIV-RNA could be undetectable [76].It seems reasonable, then, to defer PrEP start and retest after 2-4 weeks with both a fourth-generation antigen/antibody assay and HIV-RNA NAAT in case of very recent atrisk exposure or onset of AHI clinical features.Although highly sensitive, HIV-RNA testing is more expensive and not fully free from false positive results, which are likely to be the consequence of a laboratory error [77,78].In such cases, low viral loads are usually detected, and repeating HIV-RNA NAAT on a new plasma sample is recommended by US Public Health Service guidelines in the case of viral loads below 200 copies per milliliter [72].Since the occurrence of false positives cannot be excluded, we also suggest that a low-level viremia on a single plasma sample, without a complementary positive fourth-generation test, should prompt confirmation with an HIV-RNA NAAT on a further plasma sample (Figure 4).Although there is a potential greater risk for NRTIs Resistance-Associated Mutations (RAMs) insurgence with the use of self-prescribed PrEP in unrecognized HIV infections, a recent experience highlighted how the consistent implementation of self-testing in this population overcame the risk for RAMs selection in community-based services.Given this, none of the included studies reported a breakthrough HIV infection during selfprescribed PrEP [79].Although there is a potential greater risk for NRTIs' Resistance-Associated Mutations (RAMs) insurgence with the use of self-prescribed PrEP in unrecognized HIV infections, a recent experience highlighted how the consistent implementation of self-testing in this population overcame the risk for RAMs selection in community-based services.Given this, none of the included studies reported a breakthrough HIV infection during self-prescribed PrEP [79].

How to Manage Breakthrough AHI
Quarterly HIV testing is required for all PrEP users, which allows for timely diagnosis and treatment of breakthrough HIV infections.However, there is increasing evidence that exposure to antiretrovirals used as PrEP at the time of infection may alter the dynamics of viremia and the patient's immune response [80].Indeed, AHI in PrEP users usually presents with a lower viral load peak and set point and a prolonged seroconversion period, which might be delayed by several weeks.Antibody development may occur out of synchrony, with detectable antibodies in the absence of detectable antigens.Consequently, HIV testing during follow-up visits may yield ambiguous results, which, although infrequent, may be responsible for either delay in accurate diagnosis (falsely negative) or psychological stress (falsely positive) [81].
Because ambiguous results are often due to very early infection or technical issues, a reasonable strategy to confirm the presence or absence of infection is repeat testing in a few days or weeks [81].
Some authors recommend adherence-driven management of patients while their HIV status is being confirmed; if adherence is high, continuing PrEP may be the best decision, whereas in the case of inconsistent adherence, a timely transition to ART may be reasonable [81].These strategies are based on the supposed pre-test probability of AHI according to different levels of adherence to PrEP, but they are both characterized by ongoing antiretroviral use, which could suppress viral replication, thus making HIV diagnosis more difficult [81].Moreover, continuous antiretroviral exposure and limited viral replication would make it arduous to perform a genotypic resistance testing, which is essential in the subsequent management of breakthrough AHI.This appears even more true if we consider the significant rate of transmitted drug resistance observed in previous studies.
Testing for HIV-DNA might be helpful, but its use is currently limited to the research field at selected laboratories.Furthermore, it is known that viral seeding of peripheral blood mononuclear cells (PBMCs) occurs in the earlier stages of infection, and this phenomenon could be hindered by early exposure to antiretrovirals [82].Consequently, HIV-DNA assays may yield false negative results in AHI in people taking PrEP.
In this context, discontinuation of PrEP for 1-2 weeks may be resolutive, allowing for viral replication in infected patients.This approach, though operationally simple, has two main pitfalls.First, if the patient is uninfected, there is a higher risk of infection in the case of ongoing sexual risk exposure; second, in the case of unrecognized infection, there is a reduction in viro immunological benefits related to rapid diagnosis and treatment, as well as in prevention benefits against onward transmission [81].However, we believe that the transient use of other HIV-prevention strategies (condom, sexual abstinence), along with appropriate counselling, would reduce both the risk of infection in uninfected patients and the risk of onward transmission in patients with unrecognized AHI.PrEP discontinuation in infected patients would also allow for a sufficient viral replication to perform genotypic resistance testing, which would guide future treatment choices.
According to BHIVA/BASHH guidelines, TDM for tenofovir and emtricitabine should be considered to assess adherence [73].However, TDM only offers insight into recent dosing, and it does not provide reliable information about cumulative dosing adherence, which can be evaluated through more advanced but costly and poorly available methods of drug analysis (tenofovir diphosphate level in red blood cells on DBS, hair analysis and segmental hair analysis) [11].Nonetheless, despite providing a better understanding of the mechanisms underlying AHI during PrEP, pharmacology alone is currently insufficient to inform treatment decisions.
Once HIV infection is established, there are three key factors to be considered, namely timing of acquisition, level of adherence, and risk of resistance mutations.If HIV infection occurs before PrEP start, the risk of drug resistance is the highest, while it is generally lower when HIV infection is acquired after PrEP start.In the setting of optimal adherence to PrEP, breakthrough infections are extremely rare and usually associated with PrEP-resistant HIV strains [66][67][68][69][70][71].Conversely, in the setting of poor adherence to PrEP, HIV infection is more frequent, but drug resistance is uncommon because of insufficient drug pressure [80].
Regardless of the specific pathway of infection, which is difficult to identify in daily clinical practice, HIV treatment should be initiated while waiting for the results of baseline resistance testing.Most guidelines recommend upgrading the TDF/FTC PrEP regimen to a three-drug regimen by including a third drug with a high genetic barrier to resistance (dolutegravir, bictegravir, or boosted darunavir) [72][73][74][75].Despite limited evidence, such regimens are preferred as they seem to be effective even in the case of drug resistance mutations (M184I/V and K65R, conferring resistance to FTC and TDF, respectively).Twodrug regimens, such as dolutegravir plus lamivudine, are currently not recommended for first-line therapy, as data on breakthrough infections are lacking [83].

Future Perspectives in the Era of Long-Acting PrEP
Recently, new PrEP options with novel administration modalities have been proposed in an attempt to overcome adherence issues in people struggling with daily pill regimens [84].Cabotegravir long-acting (CAB-LA), given as intramuscular injections every 2 months, proved to be superior to oral PrEP in different target populations and was approved by the US Food and Drug Administration in December 2021 [62,85].
However, some concerns have been raised about HIV infections acquired during CAB-LA PrEP or before its start but unrecognized.Although rare, new HIV infections in this setting have a different clinical and virological presentation from AHI, which is often symptomatic and readily detectable with traditional laboratory assays.Longacting early viral inhibition (LEVI) syndrome is the term coined to describe the unique characteristics of new HIV infections in the setting of CAB-LA PrEP, including smoldering viral replication, delayed detection with traditional fourth-generation assays, increased risk of drug resistance, and minimal or no symptoms [62].The observed increased risk of resistance to integrase-strand-transfer inhibitors (INSTI) is particularly troubling, as most international guidelines recommend INSTI-based regimens as the first-line treatment for new HIV infections [8,74,75,86].
In this uncharted scenario, HIV screening strategies should be adjusted, and consistent evidence supports the US Public Health Service guidelines, which recommend the use of HIV-RNA testing within one week before starting CAB-LA PrEP, at every injection visit, and quarterly for 12 months after stopping injections [72].Sensitive RNA assays, indeed, detect most new infections before major INSTI resistance mutations develop [87].However, the cost-effectiveness of HIV-RNA testing is yet to be determined, and the limited availability and higher costs of this screening strategy would probably prevent wide access to CAB-LA PrEP, especially in low-and middle-income countries.It is noteworthy that in the setting of CAB-LA PrEP implementation without NAAT, the predicted higher risk of INSTI resistance should be balanced with the significant decline in new HIV infections [88,89].
Prolonged screening after injection discontinuation is required because of the risk of drug-resistant HIV infection during the so-called "tail period", during which cabotegravir plasma concentrations drop under protective levels, though maintaining potential for selective pressure.For the same reason, people with ongoing risk of HIV exposure should be offered daily oral PrEP during this time [84].
As for the treatment of new HIV infections, US Public Health Service guidelines suggest avoiding INSTI-based regimens and recommend the initiation of a three-drug regimen with boosted-darunavir, pending the results of baseline genotypic resistance testing [8].
Further studies are still needed to evaluate the feasibility of HIV-RNA screening on a large scale and to identify optimal treatment regimens for breakthrough infections.

Figure 1 .
Figure 1.Study identification and selection process.

Figure 1 .
Figure 1.Study identification and selection process.

Figure 2 .
Figure 2. Rate of seroconversions and true breakthrough infections among oral PrEP users in clinical trials.

Figure 2 .
Figure 2. Rate of seroconversions and true breakthrough infections among oral PrEP users in clinical trials.

Figure 3 .
Figure 3. Decision algorithm for PrEP start in the absence of risk factors for recent HIV infection.POC: point-of-care.

Figure 3 .
Figure 3. Decision algorithm for PrEP start in the absence of risk factors for recent HIV infection.POC: point-of-care.Viruses 2024, 16, x FOR PEER REVIEW 13 of 20

Figure 4 .
Figure 4. Decision algorithm for PrEP start when at least a risk factor for recent HIV infection is present.Of note, a positive HIV-RNA NAAT with a number of copies per milliliter close to the limit of detection should be interpreted cautiously, as false positive cannot be excluded; in such cases, a confirmatory HIV-RNA on a new plasma sample should be ordered.

Figure 4 .
Figure 4. Decision algorithm for PrEP start when at least a risk factor for recent HIV infection is present.Of note, a positive HIV-RNA NAAT with a number of copies per milliliter close to the limit of detection should be interpreted cautiously, as false positive cannot be excluded; in such cases, a confirmatory HIV-RNA on a new plasma sample should be ordered.

Table 1 .
Case reports reporting breakthrough HIV infection in PrEP users.

Table 1 .
Case reports reporting breakthrough HIV infection in PrEP users.

Table 2 .
Observational studies reporting breakthrough HIV infection in PrEP users.

Table 3 .
Randomized clinical trials reporting breakthrough HIV infection in PrEP users.