Factors associated with low-level viraemia in people with HIV starting antiretroviral therapy: A Swedish observational study.

OBJECTIVE: Low-level viraemia (LLV) occurs in some people with HIV (PWH) receiving antiretroviral therapy (ART) and has been linked to inferior treatment outcomes. We investigated factors associated with LLV in a nationwide cohort of Swedish PWH starting ART.
METHODS: Participants were identified from the InfCareHIV register, with the following inclusion criteria: ART initiation 2006-2017, age >15 years, ≥4 viral load (VL) results available and no documented treatment interruptions or virologic failure (≥2 consecutive VL ≥200 copies/ml) during follow-up. Starting from 6 months after ART initiation, participants were followed for 24 months and categorised as viral suppression (VS; VL <50 copies/ml) or LLV (≥2 consecutive VL 50-199 copies/ml). We analysed the association between the following factors and LLV using multivariable logistic regression: sex, age, pre-ART VL and CD4 count, ART regimen, country of birth, HIV-1 subtype and transmission category.
RESULTS: Among 3383 participants, 3132 (92.6%) had VS and 251 (7.4%) had LLV. In univariable analyses, factors associated with LLV were male sex, higher age, lower pre-ART CD4 count, higher pre-ART VL and ART regimen. After adjustment, the following factors were associated with LLV (adjusted odds ratio; 95% confidence interval): male sex (1.6; 1.1-2.3), higher pre-ART VL (2.7; 2.2-3.3), pre-ART CD4 count <200 cells/μl (1.6; 1.2-2.2), protease inhibitor (PI)-based regimen (1.5; 1.1-2.1), non-standard ART (2.4; 1.0-5.5) and injecting drug use (2.0; 1.1-3.7).
CONCLUSION: Among Swedish PWH, LLV during ART was associated with markers of HIV disease severity before starting ART, male sex, injecting drug use and use of PI-based or non-standard ART regimens.

Introduction

Although most people with HIV (PWH) receiving antiretroviral therapy (ART) achieve persistent viral suppression, some individuals have low but detectable plasma levels of HIV RNA without meeting criteria for virologic failure, a phenomenon commonly referred to as low-level viraemia (LLV) [1]. LLV could arise from two separate mechanisms: either passive release of viral particles from latently infected cells or as a consequence of ongoing replication [2-J Acquir Immune Defic Syndr. 2001 ">4]. LLV confers increased risk of virologic failure [5-9] and has also been associated with all-cause mortality in a recent analysis of the nationwide Swedish HIV cohort [10]. In order to interpret these associations, it is important to understand the factors that may influence development of LLV during ART.

Parameters reflecting HIV disease severity at ART initiation (high viral load [VL] and low CD4 count) have been associated with LLV in some studies [11-13], in which associations with sex and HIV acquisition route were also reported [12, 13]. Apart from host and viral characteristics, treatment-related factors might influence the risk of LLV, with higher risk for protease inhibitor (PI)-based regimens compared with those based on non-nucleoside reverse transcriptase inhibitors (NNRTIs) or integrase strand transfer inhibitors (INSTIs) [11, 14, 15]. However, these studies were conducted before introduction of INSTI-based regimens for first-line ART, and the potential association between ART regimen and risk of LLV thus remains uncertain for contemporary treatment practices.

Here, we aimed to investigate viral, host and treatment-related factors with regard to development of LLV in a nationwide cohort of Swedish PWH followed after ART initiation.

Materials and methods

Patient selection

We used data from the nationwide InfCareHIV register, a quality register encompassing >99% of Swedish PWH [16]. This register contains information on demographics, HIV acquisition route, CD4 counts, VL, HIV-1 subtype, antiretroviral drugs prescribed and results from genotypic resistance testing [17]. We included participants who started ART (≥3 non-booster drugs, of which not all were nucleoside/nucleotide reverse transcriptase inhibitors) January 1, 2006, to December 31, 2017, were >15 years old at ART initiation and had ≥ 4 VL measurements during the period 6–30 months after initiation of ART. Participants with documented treatment interruptions or virologic failure (defined as ≥2 consecutive VL measurements ≥200 copies/ml or any VL ≥1,000 copies/ml) during the 24 months observation period were excluded, as well as individuals with HIV-2 infection. The Regional Ethics Committee of Lund, Sweden, approved the study (2017/1023). No specific consent was deemed required for this study; data were pseudo-anonymized.

Definition of viraemia categories

Participants were divided into two categories based on their longitudinal viraemia profile during the observation period: viral suppression (VS) and LLV. VS was defined as VL <50 copies/ml and LLV was defined as ≥2 consecutive VL measurements of 50–199 copies/ml with >4 weeks interval (or one VL in the range 50–199 and one in the range 50–999 copies/ml, thus not meeting the definition of virologic failure). Persons with isolated VL measurements of 50–999 copies/ml preceded and followed by VL<50 copies/ml were included in the VS category.

Statistical analysis

The following variables were considered: sex, age at ART initiation, pre-ART CD4 count (higher/lower than 200 cells/μl) and VL (modelled logarithmically), route of transmission (heterosexual contact, male-to-male sexual contact, injecting drug use and other), country of birth (Nordic countries [Sweden, Denmark, Norway, Finland and Iceland] yes/no), HIV subtype (B/non-B) and type of initial ART regimen (NNRTI-based, PI-based, INSTI-based and non-standard ART [including regimens with more than one anchor drug or fusion/entry inhibitors]). Separately, we also considered CD4 percentage, rather than absolute count, and the CD4:CD8 ratio.

Data were analysed using binary logistic regression. Variables included in the main multivariable model were sex, age, country of birth, transmission category, pre-ART VL, pre-ART CD4 count and ART regimen. The association between HIV subtype and LLV was analysed separately, with adjustment for country of birth and transmission route, in addition to sex and age. For the multivariable analyses, correlation matrices were made with the included independent variables to check for multicollinearity. To investigate if the same factors were associated with LLV in men and women with HIV, respectively, we separately performed an analysis stratified by sex. Likewise, since the management of HIV has changed considerably during the studied years, we performed an analysis stratified by year of treatment initiation (before/after January 1, 2014, the year when dolutegravir became available in Sweden). Missing data were handled using complete case analysis, and we report the number of missing values per each variable. As a sensitivity analysis, age was also modelled using restricted cubic splines. We used IBM SPSS 27 Statistics for Windows, version 10 (IBM Corp., Armonk, NY, United States) and Stata SE/15.1 for Windows (StataCorp LLC, College Station, TX, United States) and report all odds ratios (OR) with 95% confidence intervals (CI). Fig 2 was constructed using R [18] with the ggplot2 package [19].

Results

Participant characteristics

A total of 5821 persons started ART between January 1, 2006, and December 31, 2017. Of these, 2438 (41.9%) were excluded (Fig 1). Of 251 individuals excluded since they met the definition of virologic failure during the observation period, 16 (6.4%) had LLV before reaching failure. Among the remaining 3383 participants, 3132 (92.6%) had VS, and 251 (7.4%) met the study definition of LLV. Approximately similar proportions of LLV were observed across calendar year of ART initiation (Fig 2). The median age at ART initiation was 38 years and 62.6% of the study participants were male. The most common transmission route was heterosexual contact (52.7%), and acquisition through injecting drugs was reported in 141 cases (4.2%). A majority (62.7%) of the participants had pre-ART CD4 count >200 cells/μl, and the median pre-ART VL was 4.8 log10 copies/ml (Table 1). The median number of VL measurements during the study period after starting ART was 5 for VS and 7 for LLV. The median VL during follow-up was <20 copies/ml for VS and 46 copies/ml for LLV.

Fig 1

Flow chart of study inclusion and exclusion.

Abbreviation: ART, antiretroviral therapy.

Fig 2

Proportion of study participants experiencing LLV during 6–30 months after start of ART, grouped by year of ART initiation.

Abbreviations: ART, antiretroviral therapy; LLV, low-level viraemia.

Table 1

Characteristics of study participants.

	Total n = 3383	Virological suppression n = 3132 (92.6%)	Low-level viraemia n = 251 (7.4%)
Male	2119 (62.6%)	1941 (62.0%)	178 (70.9%)
Age at ART initiation (years)	38 (32–47)	38 (31–47)	40 (33–48)
ART initiation >2014	1016 (30.0%)	940 (30.0%)	76 (30.3%)
Country of birth
Sweden	1176 (34.8%)	1083 (34.6%)	93 (37.1%)
Nordic countries	1255 (37.1%)	1158 (37.0%)	97 (38.7%)
Non-Nordic countries	2093 (61.9%)	1940 (61.9%)	153 (61.0%)
Missing	35 (1.0%)	34 (1.1%)	1 (0.4%)
Transmission category
Heterosexual contact	1783 (52.7%)	1653 (52.8%)	130 (51.8%)
Male-to-male sexual contact	1115 (33.0%)	1038 (33.1%)	77 (30.7%)
Injecting drug use	141 (4.2%)	126 (4.0%)	15 (6.0%)
Mother-child	16 (0.5%)	16 (0.5%)	0
Blood products	34 (1.0%)	34 (1.1%)	0
Other/Unknown	232 (6.9%)	206 (6.6%)	26 (10.4%)
Missing	62 (1.8%)	59 (1.9%)	3 (1.2%)
HIV-specific characteristics
Pre-ART CD4 count (cells/μl)	270 (150–390)	273 (160–400)	162 (67–260)
Pre-ART CD4 count <200 cells/μl	1070 (31.6%)	932 (29.8%)	138 (55.0%)
Missing	193 (5.7%)	181 (5.8%)	12 (4.8%)
Pre-ART CD4 percentage (%)	23 (16–30)	23 (16–30)	18 (12–25)
Missing	182 (5.4%)	168 (5.4%)	14 (5.6%)
Pre-ART CD4:CD8 ratio	0.45 (0.28–0.70)	0.46 (0.29–0.70)	0.35 (0.20–0.55)
Missing	608 (18.0%)	556 (17.8%)	52 (20.7%)
Pre-ART VL (log10 copies/ml)	4.8 (4.2–5.4)	4.7 (4.1–5.3)	5.5 (5.1–6.0)
Pre-ART VL ≥100 000 copies/ml	1255 (37.1%)	1069 (34.1%)	186 (74.1%)
Missing	210 (6.2%)	199 (6.4%)	11 (4.4%)
HIV-1 subtype
A	54 (1.6%)	50 (1.6%)	4 (1.6%)
B	801 (23.7%)	750 (24.0%)	51 (20.3%)
C	349 (10.3%)	324 (10.3%)	25 (10.0%)
CRF	538 (15.9%)	495 (15.8%)	43 (17.1%)
Other	136 (4.0%)	120 (3.8%)	16 (6.4%)
Missing	1505 (44.5%)	1393 (44.5%)	112 (44.6%)
ART regimen
NNRTI-based	1431 (42.3%)	1351 (43.1%)	80 (31.9%)
PI-based	1261 (37.3%)	1143 (36.5%)	118 (47.0%)
INSTI-based	634 (18.7%)	590 (18.8%)	44 (17.5%)
Non-standarda	57 (1.7%)	48 (1.5%)	9 (3.6%)

Abbreviations: ART, antiretroviral therapy; CRF, circulation recombinant forms; INSTI, integrase strand transfer inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor; PI, protease inhibitor; VL, viral load.

a Including regimens with more than one anchor drug (n = 54) or regimens based on the fusion inhibitor enfuvirtide (n = 1) or the CCR5 antagonist maraviroc (n = 2).

Values are No. (%) or median (interquartile range).

Factors associated with LLV

The following factors were associated with LLV in univariable analyses: sex, age, pre-ART CD4 count, pre-ART VL and PI-based and non-standard regimen (compared with NNRTI-based). When adjusting for sex, age, pre-ART CD4 count and VL, transmission category, country of birth and ART regimen, a statistically significant association to LLV was found for male sex (adjusted OR [aOR], 1.6; 95% CI, 1.1–2.3), pre-ART CD4 count <200 cells/μl (aOR, 1.6; 95% CI, 1.2–2.2), high pre-ART VL (aOR, 2.7; 95% CI, 2.2–3.3), PI-based regimen (aOR, 1.5; 95% CI, 1.1–2.1), non-standard ART (aOR, 2.4; 95% CI, 1.0–5.5) and injecting drug use (aOR, 2.0; 95% CI, 1.1–3.7) (Table 2). Modelling age with restricted cubic splines did not change these results.

Table 2

Factors associated with low-level viraemia in univariable and multivariable analyses.

	Unadjusted OR	Adjusted ORa
Male sex	1.5 (1.1–2.0)	1.6 (1.1–2.3)
Age at ART initiation	1.02 (1.00–1.03)	1.00 (0.99–1.02)
Pre-ART CD4 <200 cells/μl	3.0 (2.3–3.9)	1.6 (1.2–2.2)
Pre-ART VL (modelled logarithmically)	3.0 (2.5–3.6)	2.7 (2.2–3.3)
ART regimen
NNRTI-based (Ref)
PI-based	1.7 (1.3–2.3)	1.5 (1.1–2.1)
INSTI-based	1.3 (0.9–1.8)	1.0 (0.7–1.5)
Non-standardb	3.2 (1.5–6.7)	2.4 (1.0–5.5)
Transmission category
Male-to-male sexual contact (Ref)
Heterosexual contact	1.1 (0.8–1.4)	1.3 (0.9–1.9)
Injecting drug use	1.6 (0.9–2.9)	2.0 (1.1–3.7)
Other	1.4 (0.9–2.2)	1.7 (1.0–3.0)
Born outside the Nordic countries	0.9 (0.7–1.2)	1.2 (0.8–1.7)

Abbreviations: ART, antiretroviral therapy; INSTI, integrase strand transfer inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor; OR, odds ratio; Ref, reference; PI, protease inhibitor; VL, viral load.

a 3003 cases included in the multivariable analysis. Adjusted for the variables in the table.

b Including regimens with more than one anchor drug or regimens based on fusion/entry inhibitors.

Replacing absolute CD4 counts with CD4 percentages did not substantially change our conclusions regarding the other variables; having higher CD4 percentage was linked to lower odds of LLV (aOR, 0.98; 95% CI, 0.96–0.99). Having a low CD4:CD8 ratio was associated with higher odds of LLV in univariable analysis, but when adjusted for absolute CD4 counts and the other variables in the full model, this was not statistically significant (aOR, 0.9; 95% CI, 0.6–1.4).

In a subanalysis restricted to participants with available HIV subtype, we observed no statistically significant association between HIV subtype B (versus non-B) and the risk of LLV, neither in univariable analysis or after adjustment for sex, age, country of birth and suspected route of transmission (aOR, 0.8; 95% CI, 0.5–1.3).

Furthermore, in a subanalysis stratified by sex, the association between pre-ART CD4 count and LLV was not observed for females (Table 3). The association between higher pre-ART VL and higher odds of developing LLV was statistically significant in both men and women, and in both sexes starting ART before and after 2014. The increased odds of LLV in injecting drug users was restricted to persons initiating ART before 2014 (Table 4).

Table 3

Association between low-level viraemia and pre-ART CD4 count, pre-ART viral load and type of ART, stratified by sex.

	Females: n = 1109 Adjusted ORa	Males: n = 1933 Adjusted ORa
Age at ART initiation	1.00 (0.98–1.03)	1.01 (0.99–1.02)
Pre-ART CD4 <200 cells/μl	1.1 (0.6–1.9)	2.1 (1.4–2.9)
Pre-ART VL (modelled logarithmically)	2.2 (1.5–3.1)	3.1 (2.5–3.9)
ART regimen
NNRTI-based (Ref)
PI-based	2.2 (1.2–4.2)	1.2 (0.8–1.8)
INSTI-based	1.0 (0.4–2.5)	0.9 (0.6–1.5)
Non-standardb	5.0 (1.2–20.6)	1.5 (0.5–4.4)
Born outside the Nordic countries	1.0 (0.4–2.0)	1.3 (0.9–1.9)

Abbreviations: ART, antiretroviral therapy; INSTI, integrase strand transfer inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor; OR, odds ratio; Ref, reference; PI, protease inhibitor; VL, viral load.

a Adjusted for the variables in the table.

b Including regimens with more than one anchor drug or regimens based on fusion/entry inhibitors.

Table 4

Association between low-level viraemia and pre-ART CD4 count, pre-ART viral load and type of ART, stratified by starting ART before/after 2014.

	Before 2014: n = 2156 Adjusted ORa	After 2014: n = 847 Adjusted ORa
Male sex	1.2 (0.8–1.8)	3.0 (1.5–6.2)
Age at ART initiation	1.01 (0.99–1.02)	1.00 (0.98–1.03)
Pre-ART CD4 <200 cells/μl	1.5 (1.0–2.1)	1.9 (1.1–3.3)
Pre-ART VL (modelled logarithmically)	3.0 (2.3–3.8)	2.3 (1.7–3.2)
ART regimen
NNRTI-based (Ref)
PI-based	1.4 (1.0–2.1)	1.6 (0.7–3.6)
INSTI-based	0.5 (0.1–2.1)	0.8 (0.4–1.6)
Non-standardb	3.1 (1.2–7.8)	0.8 (0.1–7.6)
Transmission category
Male-to-male sexual contact (Ref)
Heterosexual contact	1.7 (1.0–2.6)	0.8 (0.4–1.6)
Injecting drug use	2.3 (1.1–4.6)	1.2 (0.2–6.7)
Other	1.9 (0.9–3.8)	1.4 (0.6–3.4)
Born outside the Nordic countries	1.0 (0.6–1.5)	1.6 (0.8–3.1)

Abbreviations: ART, antiretroviral therapy; INSTI, integrase strand transfer inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor; OR, odds ratio; Ref, reference; PI, protease inhibitor; VL, viral load.

a Adjusted for the variables in the table

b Including regimens with more than one anchor drug or regimens based on fusion/entry inhibitors.

Discussion

In this study, based on a nationwide cohort of Swedish PWH, LLV during ART was associated with higher pre-ART VL and lower CD4 count, as well as with male sex, injecting drug use and use of PI-based or non-standard ART regimens.

LLV, defined as VL of 50–199 copies/ml, occurred in 7.4% of participants, illustrating that this phenomenon is relatively common in persons receiving modern ART regimens. Although differences in the definition of LLV, cohort composition and follow-up time preclude direct comparisons between studies, the frequency of LLV in our cohort was comparable to contemporary reports from other high-income settings [11-13].

Our findings of associations between higher pre-ART VL and lower pre-ART CD4 (both absolute counts and percentage) and LLV are in agreement with previous studies [11–13, 15]. Both these factors reflect more advanced HIV infection, which suggests that LLV may be related to a larger HIV reservoir [20], and that passive release of virions from latently infected cells could be an important mechanism for LLV in our study population [2, 3]. Apart from these HIV-specific factors, we also observed that LLV was more common in men than in women. This finding is in line with studies conducted in Spain and Austria [12, 13]. In the Austrian study, associations with sex were stratified by transmission route, which showed that females infected through heterosexual contact had lower risk of LLV compared with men who have sex with men [13]. In line with this previous study [13], we observed higher risk of LLV in people with injecting drug use as their transmission category. However, this association was not statistically significant in the subanalysis of people starting ART after 2014. This might reflect changes in the Swedish HIV epidemic and/or better management of HIV in people who inject drugs during the study period. However, the number of people infected with HIV through injecting drug use was small (4.2% of the study population), so the findings of this subanalysis should be interpreted with caution. In our population, the association between sex and LLV was restricted to persons starting ART after 2014, although the proportions of men and women were similar during the whole study period. It is possible that the association between LLV and sex reflects differences in disease severity at HIV diagnosis between men and women, for instance related to late presentation [21].

Previous studies have found higher risk of LLV among people receiving PI-based compared to NNRTI-based ART [11, 12, 14, 15, 22]. We also observed such an association, but in the subanalyses restricted to participants starting ART before or after 2014, this relationship was not statistically significant. The association between different regimens and viraemia profile during ART might be influenced by indication bias, with higher likelihood of PI prescription in persons with anticipated lower adherence [11, 13]. Yet, the association between PI and LLV has also been observed in a retrospective analysis of two randomized trials [23] and is also supported by increased occurrence of viral blips in PWH on PI-based compared to other regimens [24]. We also observed higher odds of LLV among people with non-standard ART regimens (including combinations of more than one core agent and fusion or entry inhibitors). The number of patients with these regimens was small, and we consider it likely that they reflect a special category. For example, detection of pre-ART drug resistance mutations may have led to use of non-standard initial regimen. Some of these persons could also have undocumented previous ART experience. Bernal et al. reported lower frequency of LLV in persons receiving INSTI-based regimens [12], but in our material, we found no indication that INSTI-based ART confers reduced risk of LLV compared with other regimens.

Since 2015, ART is recommended for all PWH, irrespective of disease severity [25, 26]. In Swedish guidelines, ART has been recommended irrespective of CD4 counts since 2014 [27]. Since high pre-ART VL and low CD4 count were major risk factors for LLV in our material, earlier ART initiation could be expected to result in reduced incidence of LLV. Still, advanced disease at HIV diagnosis remains common in Europe [28], so improved testing strategies are likely needed to consolidate the effect of early ART on the occurrence of LLV on a population level.

The main limitation of this study concerns residual confounding. We adjusted for several potential confounders, but the database did not contain information on some factors which have been associated with LLV in other studies, such as socioeconomic status or treatment adherence [29]. Furthermore, misclassification of viraemia category might have occurred due to relatively infrequent sampling (reflecting clinical practice). For example, it is possible that some individuals categorized as virally suppressed may have had unrecognized shorter periods of LLV. Also, some individuals with LLV were excluded since they developed failure; our results are therefore restricted to people with LLV without treatment failure during the first years after ART initiation. Lastly, while previous data indicate that type of VL assay could be associated with LLV [13], we lack information on which assays that were used for specific VL measurements. The major strength of our study, which is the first to describe factors associated with LLV in Northern Europe, is that it is based on a nationwide cohort of PWH. Furthermore, we present characteristics of LLV among patients starting ART during a period with access to modern INSTI-based regimens.

Conclusion

Among Swedish PWH initiating ART between 2006 and 2017, markers of HIV disease severity at ART initiation were associated with higher risk of LLV. This risk was also increased in men, people who acquired HIV through injecting drug use and in patients receiving PI-based or non-standard ART regimens. Our findings imply that early initiation of ART, which is now recommended for all PWH, could result in reduced occurrence of LLV.

22 Mar 2022

27 Apr 2022

Comments of reviewer #1

• Comment 1: justify the inclusion dates of 2006-2017

Our response: Thank you for this comment. This manuscript is based on an excerpt from the InfCareHIV register restricted to persons who started ART between 2006 and 2017. More sensitive viral load assays (with a limit of quantification of ≤40 copies/ml) were increasingly in use in Sweden that year. We agree that it is important to consider the changing management of people with HIV over the study years when interpreting our results; however, we believe that this is addressed in the analysis of participants starting ART 2014 or later (the year when early ART was recommended for all people with HIV in Sweden, and when dolutegravir became available). Since we followed participants 30 months after start of ART, we were not able to include those who started ART 2018 or later.

• Comment 2: did you only include person who received 3 drug regimen

Our response: We agree that this was not clear in the previous version. We now added (line 75-76): “(≥3 non-booster drugs, of which not all were nucleoside/nucleotide reverse transcriptase inhibitors)”.

• Comment 3: it is not clear how was LLV defined- did this occur any time during the 24 months after ART initiation or at 24 months- how did you deal with persons whose viral load was still suppressing but not yet to that level

Our response: LLV could occur at any time during the observation period (6-30 months after start of ART). All participants who had ≥2 consecutive VL measurements of 50-199 copies/ml during the observation period were counted as LLV. Participants with virologic failure (≥2 consecutive VL measurements ≥200 copies/ml or any VL ≥1000 copies/ml) during the 24 months observation period were excluded, and those with isolated VL measurements of 50-999 copies/ml preceded and followed by VL<50 copies/ml were included in the suppression category.

We would like to clarify one situation which was not specified in the previous version: the classification of persons with 1 VL in the range 50-199 and 1 VL in the range 200-999 (at least 4 weeks apart). These were classified as LLV (in the same vein that a single VL <1000 copies/ml was allowed for viral suppression) if they did not meet the definition of failure during the studied period. This is now clarified (line 88-89): “(or one VL in the range 50-199 and one in the range 50-999 copies/ml, not meeting the definition of virologic failure)”.

• Comment 4: I have concerns about the persons that were excluded because of viral failure or treatment interruption- might these persons have had LLV and then failed- could there be misclassification issues

Our response: Thank you for this relevant comment; these data have now been added to the manuscript (line 120-122): “Of 251 individuals excluded since they met the definition of virologic failure during the observation period, 16 (6.4%) had LLV before reaching failure.”

For this study, we follow our participants for a limited time (30 months from ART initiation), and we argue that those who develop failure within this time frame likely represent a different entity than people with LLV without failure. Still, we agree that this is relevant to discuss further, and we added a section on this to the discussion (line 258-260): “Also, some individuals with LLV were excluded since they developed failure; our results are therefore restricted to people with LLV without treatment failure during the first years after ART initiation”.

• Comment 5: the analysis was done by drug class- not sure how much adherence was playing a role and the early signal for PI related to adverse events of the earlier agents- also did you take into consideration the number of ARV drugs- was there any signal once the single tablet regimens became standard

Our response: We agree that adherence could be an important factor, but unfortunately our material does not include any adherence data, as discussed in the manuscript (line 254-255). The early unboosted PI-based regimens with multiple doses per day and more side effects were rarely used in Sweden during the studied period (data not shown). We do not have data on coformulated tablets; thus, we cannot distinguish between single tablet regimens and other regimens. Still, it remains controversial whether single table regimens improve adherence, and whether a potential difference is clinically relevant (1). As a result, and since we did not want to include too many variables, we did not consider single table regimens in our analysis.

• Comment 6: in the same vein did the incidence of LLV decrease with calendar year with newer and more tolerable ART

Our response: Thank you for this suggestion. Following this comment, we calculated the proportion of LLV among people starting ART each calendar year. Although there is some variation, our data do not suggest a decreasing incidence of LLV during the study period, and it is possible that other factors (such as time between HIV acquisition and ART initiation and changing demographics of the Swedish HIV cohort) have a greater impact than the use of modern ART regimens. Since we found these results interesting, we decided to include them in the revised version:

o Line 116: “Figure 2 was made using R (18) with the ggplot2 package (19)”

o Line 123-124: “Approximately similar proportions of LLV were observed across calendar year of ART initiation (Fig 2).”

o Line 133-135: “Fig 2. Proportion of study participants experiencing LLV during 6-30 months after start of ART, grouped by year of ART initiation. Abbreviations: ART, antiretroviral therapy; LLV, low-level viraemia.”

o Figure 2

• Comment 7: in table 4 you have suggested that LLV is only 25% as common after 2014 and yet in the discussion state that LLV common in those with "modern" ART regimens

Our response: We now describe the frequency of LLV across calendar year in Figure 2. Table 4 is a stratified analysis where participants starting ART before/after 2014 are analyzed separately.

Comments of reviewer #2

• This is a well-written manuscript describing a well-designed retrospective study addressing the important topic of low-level viremia. The fact that the analysis is drawn from all adult patients in Sweden with HIV is a strength of the manuscript.

Our response: Thank you for your encouraging comments.

• Comment 1: Introduction: Par 1. "LLV confers increased risk of VF and has also been associated with all-cause mortality in a recent analysis of the nationwide Swedish cohort (5-10)." The references supporting the first part of the sentence should be separated from those supporting the second part of the sentence. The main finding of reference 10 should be described. Is the increase mortality a result of the LLV or a result of eventual VF?

Our response: Thank you for this suggestion; we have changed the references accordingly (line 55-57). The main finding of reference 10 is increased all-cause mortality for people with LLV (both LLV of 50-999 and 50-199 copies/ml). Of note, this is likely not a result of virologic failure, since study participants who developed failure were reclassified as high-level viraemia in that study. Having high-level viraemia (≥1000 copies/ml) was also associated with increased mortality in that study (2), as expected.

• Comment 2: The rationale for Tables 3 and 4 should be provided. Otherwise, their inclusion is confusing.

Our response: Women and men with HIV have certain demographic differences, and there are comparatively few data published on women with HIV, both regarding LLV and other areas. Therefore, we consider the analysis stratified by sex to be a strength of this study. The rationale behind Table 4 is that the clinical management of people with HIV has changed considerably during the studied period. 2014 was the year when dolutegravir was introduced in Sweden and also the year when ART was recommended irrespective of CD4 counts; accordingly, it is possible that contributing factors for LLV could be different before/after 2014. We have tried to explain this further (line 106-110): ”To investigate if the same factors were associated with LLV in men and women with HIV, respectively, we separately performed an analysis stratified by sex. Likewise, since the management of HIV has changed considerably during the studied years, we performed an analysis stratified by year of treatment initiation (before/after January 1, 2014, the year when dolutegravir became available in Sweden).”

• Comment 3: The authors don't address whether there is anyway to discern whether patients with LLV may have viremia resulting from clonal proliferation. For example is there a population of persons that have experienced prolonged VS only to have LLV then become detectable. This might suggest clonal proliferation of infected cells rather than viruses completing their replication cycle.

Our comment: Thank you for this interesting comment. To our knowledge, there is no way to distinguish between so called “monotypic” (e.g. related to clonal proliferation) and “diverse” (with signs of ongoing replication) LLV without analyzing viral sequences (or perhaps 2-LTR circles as a marker of replication). Of note, we studied LLV during the first 30 months after ART initiation. Consequently, our study is not designed to study individuals with longer periods of viral suppression before LLV.

References

1. Nachega JB, Parienti JJ, Uthman OA, Gross R, Dowdy DW, Sax PE, et al. Lower pill burden and once-daily antiretroviral treatment regimens for HIV infection: A meta-analysis of randomized controlled trials. Clin Infect Dis. 2014;58(9):1297-307.

2. Elvstam O, Marrone G, Medstrand P, Treutiger CJ, Sonnerborg A, Gisslen M, et al. All-Cause Mortality and Serious Non-AIDS Events in Adults With Low-level Human Immunodeficiency Virus Viremia During Combination Antiretroviral Therapy: Results From a Swedish Nationwide Observational Study. Clin Infect Dis. 2021;72(12):2079-86.

Submitted filename: Response to reviewers_220427.docx

Click here for additional data file.

3 May 2022

Factors associated with low-level viraemia in people with HIV starting antiretroviral therapy: a Swedish observational study

PONE-D-22-03146R1

Dear Dr. Olof Elvstam,

You and your co-authors have addressed the reviewer comments very appropriately and have provided a very high level and important manuscript.

We’re pleased to inform you that your manuscript has now been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Jonathan M Schapiro

Academic Editor

PLOS ONE

9 May 2022

PONE-D-22-03146R1

Factors associated with low-level viraemia in people with HIV starting antiretroviral therapy: a Swedish observational study

Dear Dr. Elvstam:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Jonathan Michael Schapiro

Academic Editor

PLOS ONE