Effect of Antiretroviral Therapy With Tenofovir, Lamivudine And Dolutegravir On Respiratory And Peripheral Muscles Strengh In People Living With Hiv/Aids

Background Some antiretrovirals (ARVs) cause muscle toxicity and their use has been attributed to beginning of respiratory and peripheral muscle weakness in people living with HIV/AIDS (PLWHA) on treatment. Dolutegravir (DTG) has been adopted by Brazil as a first-line regimen with Tenofovir/Lamivudine (TDF/3TC) since 2017, with low toxicity profile. Due to the short use of this regimen, we have not found data in the literature regarding its effects in the respiratory and peripheral muscles in PLWHA. The aim of this study was to compare respiratory and peripheral muscle strength before and after start of this new combined ART (TDF/3TC/DTG). Methods Descriptive, longitudinal and prospective study, observational and analytical with 41 PLWHA evaluated before the initiation of antiretroviral therapy (ART) (T0) which of these, 28 were reevaluated after more than 50 days of treatment (T1).The assessments of maximum functional capacity (six-minute walk test distance), maximal inspiratory (MIP) and expiratory (MEP) pressures and handgrip strength (HGS) were performed using standardized methods. In addition, laboratory data (CD4, CD4/CD8 ratio and viral load-VL) were collected. Shapiro-Wilk test was applied for normality while Fisher's exact test and t-test or Wilcoxon test were used for comparisons of categorical and continuous variables, respectively. Pearson or Spearman correlations were used according to data normality and p-value < 0.05 were considered significant for all analyzes. Results HGS Both MIP HGS T1.


Introduction
According to reported data by the United Nations group Program on Human Immunodeficiency Virus / Acquired Immunodeficiency Syndrome (HIV / AIDS) until 2018 there were 37.9 million people living with HIV/AIDS (PLWHA) in the world, 23.3 million with access to antiretroviral therapy (ART) (1) and, after almost four decades of the AIDS pandemic, HIV infection remains a major public health problem (2). Over the years, the scientific community has been trying to understand the effect of HIV on the infected organism, and studies have shown that PLWHA have a chronic inflammatory status caused by the virus, and this is associated with factors such as immune activation, secondary to several causes (3). This persistent immune activation increases the rate of mitochondrial apoptosis induced by pro-inflammatory cytokines and HIV itself, impairing mitochondrial deoxyribonucleic acid (mtDNA) in untreated HIV-infected individuals. On the other hand, the association of cytotoxic effects of some antiretrovirals (ARVs) with this reduction in mtDNA could contribute to mitochondrial toxicity in patients on ART. Therefore, HIV infection alone seems to affect mitochondria, contributing to the mitotoxicity observed in individuals in use of ART (4).
ART has changed the natural course of the disease, turning it into a chronic condition, and immediate initiation of treatment has been recommended for all PLWHA, independently of their clinical and / or immune stage (5) aiming not only improving the quality of life of these individuals, but also reducing the risk of virus transmission. (6).
The adherence to treatment controls viral replication, decreases immune activation and takes to preservation and / or restoration of the immune system in most patients (7,8), avoiding, then, complications of the HIV / AIDS complex, which include fatigue, disability, emergence of opportunistic infections (OIs) and muscle loss (9).
Although ARVs have short-term adverse effects, the most characteristic ones appear in the long term and are related to their mitotoxicity (8). Studies have suggested a correlation between ART and the incidence of skeletal mitotoxicity (10), peripheral muscle weakness (dynapenia) (11), inspiratory muscle weakness (IMW) (12) and expiratory muscle weakness (EMW) (13). The decline in handgrip strength (HGS) may contribute to decreased life expectancy and worse quality of life with aging (14). On the other hand, the IMW limits the volume of inhaled air before coughing, indirectly decreasing the maximal expiratory pressure (MEP), which is intrinsically linked to the ability of the respiratory muscles to maintain an effective cough. Thus, respiratory muscle weakness (RMW) is 4 correlated with the onset of respiratory infections, as impaired cough is not able to promote removal of airway secretions (13,15).

Advances have been made in combating HIV infection in recent years, especially
with improved ART (16,17) although it is believed that these new ARV drugs induces fewer metabolic disorders than older drugs such as mitotoxicity, their long-term effects are not yet completely understood (18,19). In this context emerged Dolutegravir (DTG), adopted by Brazil in January 2017, replacing Efavirenz (EFV), which was part of the old first-line scheme together with Tenofovir (TDF) and Lamivudine (3TC) (20). This new medication has a low profile of toxicity and interactions, besides having a high genetic barrier to viral resistance, which causes the virus resistance to develop more slowly (8). While EFV has a favorable toxicity profile, leading to long-term suppression of viral replication (5), DTG does this same process within four weeks of treatment (21). The new drug increases the chance of viral suppression among adults by 42% compared to previous treatment using EFZ. Since this drug was recently incorporated in Brazil, there are no studies in the literature regarding its effect on the respiratory and peripheral muscle strength of patients submitted to this treatment.

Methods
We conducted a descriptive, longitudinal and prospective observational and analytical study that aimed to compare respiratory and peripheral muscle strength before and after more than 50 days of initiation of ART with Tenofovir / Lamivudine / Dolutegravir (TDF / 3TC / DTG). It was carried out with patients from a public outpatient clinic that offers referral treatment for HIV/AIDS in the city of Recife, the capital of the state of Pernambuco, an important medical center in Northeast Brazil. All ART-naive patients aged 18 to 60 years were eligible. The sample size calculation was performed based on the first Brazilian study (13) witch assessed pulmonary function in PLWHA, finding a 23% prevalence of IMW and EMW in this population. Taking into account these observations, we used the program WinPep (22) and the following criteria were adopted: probable population of 120 patients, 95% confidence interval, totaling a minimum sample of 84 patients. However, the sample size was small due to the low average enrollment of antiretroviral-naive HIV-infected patients at the place of study and data collection period (between 4 and 5 patients per month). Thus, the recruitment was performed for convenience and consecutively and the screening through analysis of the medical records on the day before the medical appointment.

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Evaluations were performed in two stages: T0 (before ARV use) and T1 (more than 50 days of ARVs). Those who were eligible and agreed to participate were included in the study after signing the informed consent form. Patients with active opportunistic infection, neuromuscular diseases, motor or cognitive sequelae, or who refused to participate in the study were excluded. Between the first and the second evaluation, the pulmonary function test was performed (through spirometry). For the second stage of the study, those who started ARV regimen other than TDF / 3TC / DTG, given up treatment, were transferred to another place of follow-up or refused to continue the study, and those who did not have time to perform the second evaluation were excluded from the study. Therapists (ASHT) (24). Three measurements were obtained at one minute interval, the highest value was considered. Men with values below 30 kgf and women below 20 kgf were considered with dynapenia (25,26).
Respiratory muscle strengths were assessed using an analog manovacuometer Murenas ® -120/+120cmH 2 O, which assessed maximal inspiratory pressure (MIP) and MEP following internationally recommended maneuvering patterns (27). Predictive values for maximal respiratory pressures (MIP and MEP in cmH2O) were calculated by adjusting gender and age using equations proposed in a previous study (28). The lower limit of each pressure was obtained by subtracting the value obtained by the regression equation for calculating the maximum respiratory pressures from the value obtained in the lower limit 6 equation (LLE). The MIP and MEP results achieved by the subjects were considered normal when their values were within the range of the predicted and normal lower limit values, as adopted in a recent study on evaluation of respiratory function in PLWHA (13). Thus, we considered patients with IMW and / or EMW whose maximal respiratory muscle forces failed to reach the lower limit than predicted by the formula.
Functional capacity was assessed by walking distance in the six-minute walk test (6MWD) following American Thoracic Society (ATS) standards (29). The expected 6MWD was calculated on the basis of proposed formulas in a previous study (30), which take into account gender, height, age and weight, and the results were expressed in meters. The maximum distance traveled was used to assess submaximal functional capacity and was written in meters (m). Individuals who traveled the distance equal to or greater than expected, represented by the value obtained in the calculation of this formula, were considered to have satisfactory functional capacity. The test was performed twice, with a time interval of one hour between them, for learning purposes, which was considered the longest distance traveled between the two tests (29).
The immunological status of the patients was assessed by CD4 and CD8 counting, CD4/CD8 ratio, and viral load (VL), performed from the fasting patient's blood collection, where the technique used to quantify CD4 and CD8 counting (expressed in cells / mm³) was Flow Cytometry / BD Multitest / Facscalibur, and VL (copies / mL) by the HIV 1 PCR RealTime technique.
Spirometry was performed according to ATS standards (31), using a digital spirometer (MicroQuark® spirometer, Cosmed -Italy), based on the theoretical values for the Brazilian population defined by previous study (32). Three to eight trials were performed until three acceptable and two reproducible curves were obtained. Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1) and Tiffeneau Index (ratio of FEV1 and FVC) were measured, measurements recorded in absolute values and predicted percentages, and the reproducible maneuver with the highest FEV1 value was selected (31).
The revaluation at T1 was carried out in the same way as at T0, except for the general data. Only one 6MWT was performed at this time, as the patient already knew how to perform it. The TDF / 3TC / DTG regimen and treatment adherence were checked at the time of reevaluation, with sufficient adherence being taken at least 80% of the medication to achieve viral suppression and maintenance (5).
Statistical analyzes were performed using the Statistical Package for Social Science software -SPSS -for Windows version 22.0 (IBM, Chicago, IL, EUA) and GraphPad Prism® version 7.00 for Windows. Descriptive (sample characterization) and inferential statistics techniques were employed, using parametric or nonparametric tests for data analysis, depending on their distribution. The Shapiro-Wilk test was applied for normality and the results presented as mean ± standard deviation or median with interquartile range (25% -75%), according to their distribution, as well as percentage values.
Categorical comparisons were performed by Fisher's exact test and continuous comparisons by t-test or Wilcoxon test. Pearson or Spearman correlations, according to data normality, were performed to evaluate possible associations. The p-value < 0.05 was considered significant for all analyzes.

P a t i e n t s f o l l o w -u p :
Sixty-five patients were eligible, of which 24 were excluded (seven were younger than 18 years old, three were older than 60 years old, two had OI, one had a neurological problem, 12 refused to participate in the study and five were not evaluated by the researcher). Thus, 41 patients were included for evaluation at T0. Of these, 23 performed spirometry, but one of them failed to reproduce acceptable and reproducible curves, even after eight attempts, thus not having their examination recorded for analysis. Respiratory disorder was found in one patient by analyzing the Tiffeneau Index, classifying it as mild obstructive disorder. There was a 36.6% loss of patients during the follow-up study (four started another ARV regimen, four refused to continue the study, one abandoned treatment, one was transferred to another medical center and three did not have time enough to perform the second assessment), so there were evaluated 28 patients in T1.

S a m p l e C h a r a c t e r i z a t i o n :
In T0, 41 patients were evaluated, with a mean age of 31.6 ± 6.5 years, 80.5% male. Only 29.3% had tertiary education and almost 30% of participants were unemployed. Regarding the practice of regular physical activity 76% of the patients reported physical inactivity, 39% were overweight and 15% with controlled hypertension, and most denied the habit of smoking. Patients who were reevaluated reported not having changed habits such as physical activity and smoking.      Tables 3 and 4. When respiratory and peripheral muscle strengths were compared before and after more than 50 days of ART treatment, there was an increase in both MIP (p = 0.0176) and HGS (p = 0.0018) ( Table 6). Regarding education, 65.9% of the subjects had studied until the end of high school. In addition, Brazil has been showing a reduction in the annual number of AIDS cases since 2013, however, the North and Northeast regions showed a growing trend in detection and in our study we detected the frequency of 39% of cases.
Most of our sample reported physical inactivity, and those who practiced physical activity maintained their activities during the period between T0 and T1, so this factor should not have influenced the outcomes of the outcomes found in our study. Those who were physically active had better muscle strength scores when compared to sedentary ones. A reasonable explanation for the association between HGS and inflammation is that muscle mass is a predictor of higher HGS and those who practice regular exercise tend to have higher muscle mass (35). Moreover, only 39% of the subjects were overweight. It is worth considering that, although the association between BMI and muscle strength was 13 not measured in the present study, changes in body fat in PLWHA are associated with increased systemic inflammation and increased mortality (36).
The high prevalence of smoking among PLWHA is known when compared to the general population (5). Our sample consisted of 47.8% smokers and / or former smokers, although there was a presence of respiratory disorder in only one (4.3%) of the patients submitted to pulmonary function test. Emerging data suggest that there is a 34% prevalence of respiratory changes in PLWHA in the pre-ART era (37) 14 The assessment of HGS has a good correlation with measurements of muscle function, such as knee extension and peak forced expiratory volume (43) (45), as found in a previous study (37). Our analyzes were not stratified by gender, as only 19.5% of our sample consisted of women.
Some authors believe that in PLWHA systemic biochemical abnormalities may occur, resulting in inspiratory muscle dysfunction, with weakness of this musculature (12).The initial evaluation of our study found that 31.7% of patients had IMW and / or EMW, with lower than expected MIP and MEP values, respectively. MEP is intrinsically linked to respiratory muscle capacity, and reduced values of this pressure imply various physical and pulmonary conditions, including neuromuscular disorders (13).
In the light of the results of previous studies, which indicated that HGS measurements would not be an adequate substitute for well-controlled clinical performance testing in PLWHA. (46), we performed the 6MWT assessment in order to obtain more robust data regarding the functional capacity of this population. In the evaluation performed before starting treatment for infection, 95.12% of the subjects did not reach the predicted 6MWD, corroborating a study in antiretroviral-naive HIV-infected patients, which also found lower than expected 6MWD values in both genders (37). Our results also support those obtained in a study conducted with PLWHA, which observed an influence of CD4 lower than 200 cells / mm³ on inspiratory muscle strength and 6MWD (12). Like these authors, we agree that this is happens due to HIV infection, because the patients in our study had high viremia at the time of the first assessment, and HIV alone could have a negative influence on functional capacity in these individuals. It is also 15 noteworthy that the high frequency of dynapenia may explain non-compliance with the 6MWD, as reduced muscle strength may contribute to the feeling of fatigue and functional limitation (47).
Of the PLWHA evaluated at T0, 61% had no immunosuppression, in other words, had CD4 count above 350 copies / mm³, and this showed a positive correlation with HGS.
So all patients without compromised immune system presented higher values of HGS.
These findings contradicted those found in the Multicenter AIDS Cohort Study (MACS) substudy (36), an American cohort that evaluated for 30 years (1994-2014) a large population of HIV-infected gay and bisexual men, where no association was found between HGS and current CD4 or nadir CD4. Additionally, another study conducted in Africa also found no independent effects between CD4 count and HGS in a cross-sectional analyzes (40). On the other hand, considering that a previous study found concentrations of Interferon Gamma-Induced Protein 10 (IP-10), an immune activation biomarker, associated with low CD4 levels (48), we suggest that the best HGS values found in the higher CD4 patients would be due to the lower inflammatory status in these patients. However, due to the absence of inflammation markers collection at the time of the evaluations, we cannot conclude the existence of this association.
HGS values obtained in the evaluation before the initiation of ART showed a negative correlation with VL. In this way, lower HGS values correlated with high levels of VL. Given these observations, we hypothesized that this reduction in HGS would be associated with the inflammatory status caused by high viremia. This hypothesis is supported by a previous study, which found a relationship between IP-10 concentrations with high VL, even suggesting that this biomarker may be associated with HIV pathogenesis and immune depletion (48). In addition, our findings also corroborate those found in the substudy, suggesting that VL cumulative increased exposure seems to be an important factor in the decline in HGS, and further highlights the importance of early initiation of ART (14).
Considering reduction in muscle strength may contribute to the feeling of fatigue and functional limitation (47), HGS provides data not available from routine clinical and laboratory evaluations. Thus, the measurement of this variable during follow-up with the infectologist may provide additional information for risk stratification during the evaluation of outpatient PLWHA.
In our study, the frequency of RMW in antiretroviral-naive HIV-infected patients was greater than 30%, however, this weakness did not correlate with CD4 and CD8 count, CD4/CD8 ratio, and VL. This results may have been influenced by the small sample size.

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MIP measurement is earlier than changes in lung volume to diagnose inspiratory weakness. MEP has a low accuracy to predict coughing capacity, as it generates a high false positive index for EMW, and may overestimate the number of patients with EMW, since low values are caused by submaximal efforts or leakage around the mouthpiece, which is frequent in patients with facial muscle weakness. Both depend on the coordination between the evaluated and the examiner, so a low value may not mean weakness, but lack of collaboration of the evaluated subject (49). We believe that this did not interfere with the results of our study, since 65.9% of the sample had a medium level of education, having completed at least high school, obtaining a good understanding of the commands given to perform the tasks evaluations, besides that the vast experience of the examniner's in such evaluations.
Newer medications tend to cause less mitotoxicity. Thus, the incidence of this toxicity may be decreasing even as more patients are submitted to treatment with Reverse Transcriptase Nucleoside Inhibitors (NTRs) such as Tenofovir and Lamivudine (50). Furthermore, studies indicate that DTG is a useful drug in terms of rapid virologic suppression in patients with good treatment adherence (21), showing superiority over the previous regiment containing EFZ Our results showed a significant increase in MIP and HGS after more than 50 days of ART, presumably through control of viral replication and subsequent reduction of inflammation and immune activation, as more than 80% of patients progressed to undetectable VL in this period. This effect is hypothesized to be mediated by ART's ability to reduce inflammation, immune activation, and endothelial dysfunction by suppressing HIV replication (7). Conclusive findings in previous research support our theory. The English Longitudinal Study of Ageing (ELSA), conducted in England with an eight-year follow-up showed that higher HGS would be associated with lower levels of inflammation (35). In another interesting study of hospitalized patients with and without TB, proved that higher levels of Tumor Necrosis Factor Alpha (TNF-α) but not Interleukin-6 (IL-6) were associated with a reduction in MIP, MEP and HGS (47). In addition, previous study data pointed out that HGS appears to steadily increase over time for ART in men, although it has stabilized after the first 12 weeks of treatment in women (40), corroborating with our findings.
Notably, the loss of 36.6% of patients during follow-up associated with the short interval between evaluations denotes a major limitation of our study. The loss to follow-up of HIV infected patients is a problem that is frequently reported on in the literature (51) and even registered in the HIV-Brazil Cohort, with the highest proportion of loss (50.4%) in the first year of follow-up, identifying the Northeast as the second largest region of loss of follow-up of patients (52). The fact that we conducted our study at a single state referral center for HIV treatment may have caused some bias. Similarly, the decrease in sample size over time limits our statistical power, although it is highly relevant, since most patients had dynapenia, even without major functional impairment. We found important, although weak, correlations between HGS and CD4 count and VL, but the lack of collection of immune activation markers such as IL-6, TNF-α and C-reactive protein (CRP) at T0 and T1 prevented the analysis of the inflammatory status of the patients at the time of the evaluation and it was not possible to establish a causal relationship between these variables. The instrument used to evaluate MIP and MEP, the analog manovacuometer, was limited to maximal pressures of 120 cmH 2 O and could have underestimated our results.
However, the cost, availability, and ease of use may determine whether the techniques are best suited to clinical practice or useful for research (26). In this context, we chose to use a lower cost instrument in order to try to reproduce as much as possible the reality of the public hospital service.