Diagnostic and Therapeutic Perspectives on Persistent HIV-1 Low-Level Viremia

Abstract

While modern antiretroviral therapy achieves high rates of virologic suppression, persistent low-level viremia (pLLV) remains a common clinical scenario that poses distinct diagnostic and therapeutic dilemmas. This review proposes a systematic diagnostic approach to pLLV, beginning with a thorough evaluation of adherence, potential drug-drug interactions, dietary considerations, and resistance profiles. Such a structured assessment is essential to differentiate diverse pathophysiological conditions, particularly in distinguishing active replication-driven failure from reservoir-derived non-suppressible viremia (NSV). Management strategies are tailored to these findings: extrinsic factors, such as suboptimal adherence or pharmacological barriers, are managed through targeted optimization, while intrinsic resistance necessitates switching to fully active regimen. In contrast, NSV is a reservoir-driven process that does not necessitate ART regimen intensification or modification. We also examine key considerations for recently introduced long-acting injectables in the context of pLLV. Furthermore, we address sexual transmission risks, emphasizing that while the risk remains extremely low within the pLLV range, clinical vigilance is crucial given the risks of virologic failure. Successful pLLV management therefore relies on a tailored approach that balances proactive clinical intervention with judicious observation, ensuring virologic control and patient quality of life.

Introduction

The primary goal of modern antiretroviral therapy (ART) is to achieve and maintain viral suppression, typically defined as a plasma human immunodeficiency virus (HIV)-1 RNA level below the limit of detection [1]. However, despite the high potency of contemporary regimens, clinicians frequently encounter patients with measurable low-level viremia (LLV) [2]. This phenomenon presents a distinct challenge in routine practice. For clinicians, it creates a diagnostic and management gray zone complicating the decision between maintaining the current regimen and switching to alternatives [3]. Concurrently, for patients, the ongoing detection of viremia often raises significant anxiety regarding treatment failure and transmission risks [4].

In clinical practice, the majority of isolated LLV episodes are virologic blips [5,6]. However, a significant clinical challenge arises when LLV remains persistent (pLLV), defined as viral loads that persist on multiple occasions between 50 and 1000 copies/mL [1,2,7,8,9]. Determining the underlying etiology of pLLV is essential to guide clinical management [2,3]. These pathophysiological drivers range from active viral replication, driven by suboptimal adherence or emerging drug resistance, to the passive release of virions from stable cellular reservoirs, a distinct subset often termed non-suppressible viremia (NSV) [10,11,12,13].

These divergent mechanisms determine therapeutic responses: while some patients achieve suppression through behavioral or ART regimen modifications, others experience NSV, a condition in which pLLV persists despite ART modification or intensification and no obvious signs of suboptimal ART adherence or drug resistance [14,15]. Furthermore, in the era of two-drug regimens and long-acting injectables, the clinical significance of pLLV has gained renewed attention, as the safety margin for resistance may differ from traditional three-drug oral therapies [16,17].

This review aims to provide a clinically actionable framework for the management of pLLV. We outline a structured diagnostic approach for differentiating pLLV from virologic blips and identifying its underlying etiology. Additionally, we evaluate therapeutic strategies tailored to these distinct virologic entities, contrasting the necessity of regimen modification in active failure with the rationale for regimen preservation in NSV. We also examine the nuanced role of long-acting injectables and the latest transmission data. By integrating these perspectives, we aim to help clinicians balance rigorous virologic vigilance with the restraint needed to prevent unnecessary regimen changes.

Diagnostic perspective

1. Normal viral decay kinetics and defining pLLV

The elimination of HIV-1 RNA from plasma following the initiation of ART follows a multi-phasic decay model [18,19]. The initial phase, occurring within the first two weeks, is characterized by a rapid viral decline with an initial decay half-life of approximately 1 to 2 days, reflecting the clearance of short-lived, productively infected CD4⁺ T cells. This is followed by a slower second-phase decay from weeks 2 to 14, representing the loss of longer-lived cellular compartments such as infected macrophages and partially activated T cells. Finally, the decay kinetics enter a long-term plateau. Using ultra-sensitive single-copy assays, Palmer et al. demonstrated that this late-stage residual viremia exhibits an extremely slow decay or steady state. This viremia originates from the stable latent reservoir and can persist even after 7 years of suppressive ART [20].

Building upon this kinetic model, defining pLLV requires careful consideration of both the timeframe and the virologic threshold. Virologic suppression is generally expected within 6 months of initiating potent ART [1,7]. However, applying a strict timeline requires caution to avoid misclassifying slow responders as having true pLLV. For instance, large cohort analyses have demonstrated that individuals with a high baseline viral load (>100,000 copies/mL) or advanced immunosuppression may require up to 48 weeks to achieve full virologic suppression [21,22,23]. Prematurely categorizing these delayed responders as experiencing virologic failure could lead to unwarranted regimen modifications, compromising future therapeutic options [21].

pLLV is generally defined as persistent detectable viremia between 50 and 1,000 copies/mL; temporally, "persistent" has usually been defined in studies as at least two consecutive detectable measurements [21,24]. Although specific time intervals between these tests are rarely defined in the literature, clinical practice typically follows major virologic monitoring guidelines, which recommend a retest interval of 2 to 4 weeks for confirmation [7,8,9]. In terms of viral load, the precise threshold remains highly heterogeneous across global guidelines, complicating clinical management [25] (Table 1). The United States Department of Health and Human Services defines virologic failure as a confirmed RNA level ≥200 copies/mL, effectively leaving the 50–199 copies/mL range as a diagnostic grey zone [7]. In contrast, while the European AIDS Clinical Society shares the goal of strict viral suppression (<50 copies/mL), its guidelines recommend more proactive interventions even within this lower-level viremia range [8]. Meanwhile, the World Health Organization (WHO) utilizes a 1,000 copies/mL cutoff for virologic failure, although recent updates mandate enhanced adherence counseling and shortened monitoring intervals for the 50–999 copies/mL range to prevent definitive failure [9]. In this review, we will discuss pLLV within the 50–1,000 copies/mL range. This broader scope encompasses the WHO guidelines and accounts for NSV cases that can persistently manifest within the 200–1,000 copies/mL range [10,11,12,13]. Furthermore, LLV below 1,000 copies/mL often presents a distinct clinical conundrum for the clinician and patient, as standard resistance testing may fail and transmission risk remains low [4,26,27]. However, we fully acknowledge that a viral load ≥200 copies/mL should be primarily considered as active virological failure, and we will explore a step-by-step diagnostic approach to address this.

Table 1. Virologic thresholds and clinical management strategies for persistent low-level viremia across major international guidelines.

Guideline/organization	Target for viral suppression	Definition of virologic failure	Concept of pLLV	Clinical approach to pLLV
US DHHS	<50 copies/mL	Confirmed HIV RNA ≥200 copies/mL	Persistent detectable HIV RNA between 50 and 199 copies/mL	Do not change regimen immediately. Check adherence, DDI, and repeat testing.
EACS	<50 copies/mL	Confirmed HIV RNA ≥50 copies/mL	Not distinctly defined as a separate entity	Proactive intervention: evaluate adherence; attempt genotypic resistance testing if possible; consider regimen switch if HIV RNA >200 copies/mL.
WHO	<50 copies/mL	Confirmed HIV RNA ≥1,000 copies/mL	Persistent HIV RNA within the 50–999 copies/mL range	Enhanced monitoring: provide enhanced adherence counseling and repeat viral load testing. Switch only if ≥1,000 copies/mL.

pLLV, persistent low-level viremia; US, United States; DHHS, Department of Health and Human Services; HIV, human immunodeficiency virus; DDI, drug-drug interaction; EACS, European AIDS Clinical Society; WHO, World Health Organization.

2. The first step: excluding non-adherence and pharmacological issues

Even in the era of highly potent modern ART, suboptimal adherence remains one of the most frequent and critical driver of pLLV [28,29]. However, relying solely on self-reported adherence in the clinic is inherently limited, as many patients tend to overestimate their compliance [30,31]. Studies utilizing objective adherence measures, such as unannounced pill counts, have demonstrated that even minor decreases in adherence, which often occur despite self-reports of 100% compliance, are significantly associated with an increased risk of pLLV [32,33]. However, these latter studies were performed in the era before highly-active modern integrase strand transfer inhibitors, such as dolutegravir and bictegravir.

Given the impact of suboptimal ART adherence, therapeutic drug monitoring (TDM) serves as a valuable objective diagnostic tool. TDM frequently reveals sub-therapeutic trough concentrations, which can differentiate unrecognized non-adherence from pharmacokinetic variability driven by altered gastrointestinal absorption or genetic polymorphisms in drug-metabolizing enzymes [28,34].

Additionally, there are a number of other causes for low ART drug levels. These include poor gastrointestinal absorption, drug-drug interactions, and the impact of body composition on long-acting ART injections. Dietary interactions must also be carefully examined. Administering rilpivirine in a fasting state reduces its area under the curve (AUC) by 43% compared to administration with a standard meal, highlighting the risk of unintentional non-adherence from dietary habits [35]. A careful review of potential drug-drug interactions is also essential, as the magnitude of these interactions is frequently underestimated in clinical practice [36,37]. For instance, simultaneous administration of dolutegravir with common polyvalent cations, such as aluminum- or magnesium-containing antacids, reduces its AUC by approximately 74%, drastically compromising efficacy [38]. Strong CYP3A4 inducers (e.g., rifampin) can also substantially reduce plasma concentrations of several ART classes. Given the complexity of modern polypharmacy, recognizing these pharmacological requirements is fundamental. Table 2 summarizes frequently encountered interactions prioritized by their potential for severe virologic consequences, such as those categorized as Red (contraindicated) or Orange (requiring significant adjustment) by the University of Liverpool database. Additionally, we highlight mandatory dietary requirements essential for ensuring adequate systemic exposure [7,39]. Furthermore, for long-acting ART cabotegravir and rilpivirine, it is critical that the dose be injected intramuscularly. High body mass index may lead to an increased risk of injection into subcutaneous fat and insufficient needle lengths may lead to suboptimal drug levels [40].

Table 2. Representative drug and dietary interactions compromising virologic efficacy in modern antiretroviral therapy.

Category	Interacting agent	Affected ARV	Impact on exposure	Clinical recommendation
Drug interaction	Antacids (Al, Mg, Ca), multivitamins, iron	INSTIs (DTG, BIC, RAL)	↓AUC by 50–74%	Separate dosing (2h before or 6h after) Avoid simultaneous intake
	PPI	ATV, RPV	↓Absorption (pH dependent)	Avoid PPI with RPV. Use H2RA with caution and specific timing
	Rifampin, St. John’s Wort, anticonvulsants	Most ARVs (PIs, INSTIs, NNRTIs)	Critical ↓AUC	Contraindicated or requires significant dose adjustment (e.g., DTG BID)
Food interaction	Fasted state (No food)	RPV, DRV/c, DRV/r, EVG/c, ATV	↓AUC by 30–50%	Must be taken with a standard meal (approx. 400–500 kcal) to ensure absorption
	High-fat meal	EFV	↑Absorption / toxicity	Take on an empty stomach to reduce CNS side effects

ARV, antiretroviral agent; INSTIs, integrase strand transfer inhibitors; DTG, dolutegravir; BIC, bictegravir; RAL, raltegravir; AUC, area under the curve; PPIs, proton pump inhibitors; ATV, atazanavir; RPV, rilpivirine; H2RA, histamine H2-receptor antagonist; PIs, protease inhibitors; NNRTIs, non-nucleoside reverse transcriptase inhibitors; BID, twice daily; DRV/c, darunavir/cobicistat; DRV/r, darunavir/ritonavir; EVG/c, elvitegravir/cobicistat; EFV, efavirenz; CNS, central nervous system.

Taken together, the initial diagnostic approach to pLLV must be anchored in a rigorous evaluation of all extrinsic factors that compromise systemic drug exposure. While TDM serves as the definitive objective tool for identifying these deficits, its routine clinical application is frequently hindered by high costs, delayed turnaround times, and a limited availability [41,42]. Therefore, providers must rely on a structured, pragmatic approach to pharmacological examination, integrating non-judgmental adherence assessments with a meticulous review of potential drug-drug and drug-food interactions (Fig. 1).

Figure 1. Integrated diagnostic and therapeutic framework for persistent HIV-1 low-level viremia.

HIV, human immunodeficiency virus; TDM, therapeutic drug monitoring; GSS, genotypic susceptibility score.

3. The second step: excluding antiretroviral resistance

Once extrinsic factors have been rigorously excluded, the persistence of viremia under optimal drug exposure raises the clinical suspicion of antiretroviral resistance. Because ongoing active replication with resistant virus necessitates immediate regimen modification, excluding this possibility is another critical step in the diagnostic workup.

Within the higher ranges of pLLV (>200 copies/mL), standard commercial genotypic resistance testing (GRT) can often yield successful and clinically actionable results [7,27]. In contrast, a diagnostic challenge arises in pLLV below 200 copies/mL. In this range, standard commercial GRT frequently fails due to insufficient viral RNA input, with amplification success rates decreasing significantly to 30–50% [26,27]. To overcome this technical limitation, clinicians may utilize specialized alternative assays for low-copy viremia, which employ ultracentrifugation of larger plasma volumes to enhance sequencing sensitivity, although these assays may only be available in research settings [26]. When plasma RNA remains insufficient, proviral DNA genotyping using peripheral blood mononuclear cells serves as an alternative [43]. This method can successfully yield a resistance profile in the majority of pLLV patients, revealing archived resistance mutations that may be driving the viremia even if they are not easily detectable in the circulating RNA pool. Crucially, the diagnostic power of both approaches is increasingly enhanced by next-generation sequencing (NGS) [44]. While traditional Sanger sequencing only detects majority variants comprising at least 15–20% of the viral population, NGS can reliably identify minority resistant variants down to a 1–5% threshold, providing a much higher resolution of the mutational landscape contributing to pLLV.

Once a genotype is successfully obtained, clinicians must interpret the mutational profile to calculate the genotypic susceptibility score (GSS). The GSS provides a quantitative estimate of the regimen’s overall antiviral potency by evaluating the resistance mutation profile, assigning a susceptibility score (ranging from 0 to 1) to represent the effectiveness of each individual drug, and summing these scores [45,46]. The clinical algorithm offers clear diverging pathways based on this score. A suboptimal GSS (typically <2) confirms active virologic failure driven by drug resistance, necessitating prompt regimen optimization. Notably, when interpreting results from DNA genotyping assays, clinicians must account for the fact that it may detect long-archived mutations or those present in defective proviruses that do not necessarily drive the current pLLV, while the clinical impact of minority variants identified by NGS is still being elucidated [43,44]. Conversely, preemptive drug switching in the absence of actionable mutations risks exhausting future therapeutic options if resistance detection is not attempted. Empirical modification without genotypic guidance should be reserved for those whose resistance genotyping have failed or high-risk patients with a documented history of sequential virologic failures. In such challenging scenarios, treatment decisions must be guided by the cumulative resistance profile and all past treatment exposures to ensure the inclusion of at least two fully active agents [1,7,47].

It is crucial to acknowledge that this comprehensive diagnostic algorithm is largely confined to resource-rich environments. In resource-limited settings, where the HIV burden is concentrated, standard GRT is often inaccessible. To address this gap, current WHO guidelines emphasize a public health approach, prioritizing adherence optimization and the strategic deployment of high-genetic-barrier regimens [9]. Global initiatives are now striving to integrate simplified resistance testing into these settings to ensure more equitable management and to prevent the transmission of resistant variants [48].

4. Final step: recognizing NSV

When pLLV persists despite confirmed ART adherence, optimal drug exposure, and the absence of resistance to the current ART regimen, the patient likely has the third cause of pLLV: NSV due to a hyperactive HIV reservoir [10]. In this distinct virologic state, detectable viremia reflects the passive, non-replicative discharge of virions from expanded cellular reservoirs rather than de novo replication. Recognizing this pathophysiological boundary is critical, as it prevents futile regimen modifications and helps patients avoid an unnecessary pharmacological burden.

Mechanistically, NSV is driven by the proliferation of clonally expanded CD4⁺ T cells harboring integrated proviruses [10,11]. These cellular clones act as stable viral sources, continuously releasing particles despite suppressive ART. Furthermore, these shed virions are often defective, particularly those with 5'-leader mutations, yet remain readily detectable by standard RNA assays [12]. This highlights that the presence of replication-competent plasma virions are not required in NSV as the mechanism is not active replication, but virion release from a hyperactive HIV reservoir. Recent evidence also indicates that a subset of NSV can originate from exceptionally large, non-clonal reservoirs that generate diverse viral populations [13]. Given that NSV relies on passive release rather than active replication, treatment modification or intensification will fail to reduce viremia and remains clinically ineffective.

Therapeutic perspective

1. Clinical management: tailoring the approach

Following the systematic diagnostic workup described above, clinicians can distinguish replication-driven viremia (active virologic failure) from reservoir-derived viremia (NSV), a pathophysiological distinction that forms the cornerstone of clinical management (Fig. 1). When the evaluation reveals correctable extrinsic barriers, such as suboptimal adherence, drug-drug interactions, or improper food intake, the therapeutic priority is targeted behavioral and pharmacological optimization rather than a regimen switch [7,47]. Once these reversible factors are addressed or ruled out, clinical management follows two divergent pathways based on the underlying virologic state.

For patients in whom active virologic failure is confirmed, either through uncorrectable pharmacological barriers or the detection of intrinsic resistance, regimen modification is indicated. The primary objective is to suppress viral replication before the accumulation of further mutations compromises future therapeutic options. When designing an optimized regimen, clinicians must rely on the patient’s cumulative resistance profile, integrating all historical and current genotypic data. Standard guidelines recommend that the modified regimen include at least two, and ideally three, fully active agents [1,7,47]. In the era of high-genetic-barrier ART, achieving this threshold is usually feasible and facilitates re-suppression even in heavily treatment-experienced individuals [49,50].

Conversely, when the diagnostic evaluation points to clinically suspected NSV, the clinical priority shifts from achieving viral suppression to long-term regimen preservation. In this state, aggressive ART modification or empirical intensification is of limited clinical benefit. While large-scale clinical trials specifically targeting NSV are lacking, the futility of intensification in this context is consistent with findings from landmark studies evaluating residual viremia below the limit of detection. For example, Dinoso et al. demonstrated that in patients with long-term viral suppression (<50 copies/mL), intensifying ART with additional agents did not further reduce or accelerate the decay of residual HIV-1 viremia [14]. Similarly, a study by McMahon et al. confirmed that adding raltegravir to the regimens of patients with strictly defined residual viremia yielded no significant virologic benefit [15]. We acknowledge that these studies primarily evaluated residual viremia below 50 copies/mL, highlighting the need for large-scale clinical trial data regarding ART intensification specifically for higher pLLV ranges. However, existing literature on NSV cases consistently indicates that regimen modification or intensification yields no virologic suppression [10,11,12,13]. This lack of efficacy is supported by a strong mechanistic rationale: because currently available antiretrovirals exclusively target de novo viral replication, they remain fundamentally ineffective against the passive, continuous release of virions from stable expanded reservoir cells. Given this mechanism, empirical intensification is highly unlikely to reduce viremia in these patients. Consequently, the optimal clinical approach for higher pLLV ranges should be heightened clinical vigilance without empirical ART intensification. This strategy ensures close monitoring for true active virologic failure while preventing exposure to avoidable toxicities and preserving the limited antiretroviral agents [7,47].

2. Emerging strategies: long-acting injectables

The advent of long-acting injectable (LAI) ART, primarily cabotegravir and rilpivirine (CAB/RPV), represents a paradigm shift in HIV management by dramatically reducing pill burden [51,52]. However, because pivotal clinical trials required strict viral suppression (HIV-1 RNA <50 copies/mL) prior to initiation, patients with a history of pLLV or those currently experiencing it present a clinical dilemma. When considering a transition to LAIs for individuals with a history of pLLV, the primary concern is the potential presence of archived resistance. Historical periods of uncontrolled viral replication can lead to the silent accumulation of mutations within the latent reservoir. In these scenarios, proactive genotypic testing of proviral DNA should be considered prior to LAI initiation to detect occult resistance undetectable by standard plasma RNA assays [7,53,54].

For patients currently experiencing active pLLV, the decision-making process relies heavily on the diagnostic framework established above. If viremia is driven by profound adherence barriers, LAIs may serve as an effective rescue strategy. Recent evidence, including real-world data from the Ward 86 clinic cohort [55] and the phase 3 LATITUDE (ACTG A5359) trial [56], consistently demonstrates that providing long-acting CAB/RPV is a highly effective rescue strategy for achieving and maintaining virologic suppression in patients with severe adherence challenges. Conversely, if the evaluation indicates clinically suspected NSV, the rationale for switching requires careful consideration. While an injectable regimen will not halt the passive release of virions from expanded reservoir, it may be cautiously considered to alleviate the burden of daily oral therapy, provided there is confirmed absence of pre-existing resistance [57].

Regardless of the underlying cause, the emergence of pLLV during established LAI therapy necessitates heightened clinical vigilance. Virologic failure on these long-acting regimens is of particular concern, as suboptimal drug exposure can rapidly facilitate the development of complex, dual-class resistance [58]. Consequently, if active virologic failure is detected during LAI treatment, clinicians must promptly implement a fully suppressive oral regimen to prevent further resistance accumulation [1,7].

3. The risk of transmission: reassurance and vigilance

The Undetectable Equals Untransmittable (U=U) paradigm is a profound public health milestone in modern HIV management [59]. The threshold for negligible sexual transmission risk is robustly established at <200 copies/mL, a range that encompasses the typical definition of pLLV. While the most robust evidence supporting U=U applies to viral loads <200 copies/mL, this reassurance now extends to viremia between 200 and 1,000 copies/mL, as recent evidence indicates that sexual transmission risk remains negligible at these levels [4,59]. Consequently, the WHO recognizes this range as presenting an effectively zero risk [9], supporting crucial reassurance to mitigate self-stigma and unnecessary anxiety in patients maintaining optimal adherence.

However, this public health reassurance must be carefully distinguished from clinical oversight, in that epidemiologically untransmittable pLLV is not necessarily clinically benign for the individual host. If pLLV stems from suboptimal adherence or emerging drug resistance, as opposed to NSV, the patient remains at risk for subsequent virologic failure and the progressive accumulation of mutations [24]. Specifically, viremia ranging from 200 to 1,000 copies/mL falls within the threshold for virologic failure in many guidelines, necessitating aggressive investigation to prevent further progression [7]. Therefore, clinicians must balance the need to offer firm reassurance regarding transmission risks to protect the patient's quality of life while simultaneously maintaining rigorous vigilance to identify and address any actionable causes of persistent viremia.

In conclusion, pLLV is a heterogeneous clinical condition that precludes a uniform management approach. By distinguishing replication-driven failure from reservoir-derived NSV through a systematic diagnostic workup, clinicians can avoid unnecessary regimen modifications while identifying those at genuine risk of virologic failure. As the HIV treatment landscape evolves with long-acting injectables, this tailored differentiation is critical to preserving future therapeutic options. Clinical success in pLLV management necessitates balancing rigorous virologic vigilance with evidence-based reassurance, ensuring both long-term viral control and an improved quality of life for patients.