Bone Involvement in Antiretroviral-Therapy–Naïve HIV-Infected Young Men

Background: Low bone mineral density has been described as a non–AIDS (Acquired Immune Deciency Syndrome)-related event in HIV (human immunodeciency virus)-patients but it is poorly studied in young HIV-infected men who have received no previous antiretroviral therapy. Methods: A cross-sectional study of 245 naïve-HIV-infected men over 21 and under 50 years old was carried out between January 2014 and September 2017. Results: The mean age was 36.4 years, been 68% Caucasian and 29.3% Latin American. At the time of diagnosis, 91% of patients had stage-A (median CD4+ T-cell 481cells/μL, IQR, 320–659). 10% had a count below 200 CD4 cells/μL, and 40% had a CD4/CD8 cell-count-ratio below 0.4. Regarding lifestyle and risk factors, 14.1% presented underweight, 36.1% engaged in no regular exercise, 51.9% had previous exposure to tobacco and 35.3% reported drug use. Low levels of vitamin D were seen in 87.6% of the men studied. Low BMD (Z-score<2.0) was found in 22,8% of the patients. Conclusions: We nd prevalence of bone involvement among naïve HIV-infected men under 50 years old. Further studies are necessary to evaluate if BMD assessment should be recommended in young HIV-infected patients.

Bone involvement, de ned as the presence of osteopenia or osteoporosis on a dual energy x-ray absorptiometry (DXA) scan, (3,4) has been the subject of numerous studies, and have focused on bone toxicity associated with ART.
A number of clinical trials have described reduced BMD during the rst or second year of ART, independently of the type of therapy, (5,6) in particular, tenofovir disoproxil fumarate (TDF) and protease inhibitors (PIs) have been associated with higher rates of medium-and long-term bone toxicity.(7) As a result, the primary international practice guidelines recommend performing a DXA scan in HIV-infected individuals over the age of 50 years. (1,7) The factors that contribute to bone involvement are widely known and include age, vitamin D de ciency, tobacco and alcohol consumption, a sedentary lifestyle, among others. (8,9) In addition to these factors, PLWH exhibit a marked proin ammatory state even after ART start. (10) Little is known, but it is expected, therefore that young HIVinfected individuals will have lower bone mass than similar uninfected population, even prior to initiation of ART. (11,12) The aim of this study is to assess bone involvement and risk factors that may contribute to the onset of low BMD among young HIV-infected men (under 50 years of age), who are naïve to ART.

Methods
A cross-sectional study of HIV-infected men over 21

Measurements and reference values
We gathered such epidemiologic data as age, race, and country of birth. Lifestyle-related aspects used as study variables included alcohol, tobacco, and drug consumption, physical activity, and approximate calcium intake.
Additionally, anthropometric data were collected for all patients. Blood test was done fasting, measured by Advia 2400 system (Siemens , Munich, Germany) for values related to calcium, phosphorus and vitamin D. Immunological and virologic parameters (i.e., CD4+, CD8+, and HIV-1 viral load) were measured by PCR (Roche, Basel, Switzerland).
Underweight was de ned as a body mass index (BMI) of <20 kg/m 2 . The values related to bone and mineral metabolism used were the following: 25OH Vitamin D (30-50 ng/ml), and parathormone (PTH) (10-70 pg/ml).
Patients considered smokers if they were current or past tobacco users; consumers of alcohol if their total intake was over 30 g/day; and drug users (cocaine, mephedrone, amphetamines, ketamine, GHB) if any of these drugs were taken at least once weekly. For the purposes of this study, su cient physical exercise was a minimum of 120 minutes per week. Three servings of calcium-rich foods (e.g., milk, cheese, other dairy products) daily was considered an appropriate intake. Qualitative variables were expressed in terms of frequency and percentages. Based on the results of the Kolmogorov-Smirnov test for normality, quantitative variables were measured as either mean and standard deviation or median and interquartile range (IQR). Qualitative variables were analyzed using the Chi-squared test or Fisher's exact test. Quantitative variables were compared using Student's t test. For all determinations, we used R software version 3.6.0 (R Core team (2020); R Foundation for Statistical Computing, Vienna, Austria), and statistical signi cance was set at p < 0.05.

Epidemiology and lifestyle
A total of 245 patients were included, all of whom were men. The main patient characteristics appear in Table 1. The median age of the patients was 36.4 years, 68% of whom were Caucasian (87.7% Spanish), and 29.3% Latin American. All patients had been infected with the HIV virus through sexual intercourse (MSM). At the time of diagnosis, 91% of patients had stage-A disease according to the classic CDC (Center for Disease Control) classi cation system, and the median CD4+ T-cell count was 481 cells/μL (IQR, 320-659). Ten percent of these stage-A patients had a count below 200 CD4 cells/μL, and 41% had a CD4/CD8 cell-count ratio below 0.4. (Table 1).
Regarding lifestyle patterns and risk factors associated with lower BMD (Fig 1), 14.1% presented underweight with low BMI 36.1% engaged in no regular exercise, 51.9% had previous exposure to tobacco, 35.3% reported drug use, and 9.3% drank alcohol habitually. Abnormally low levels of vitamin D were seen in 87.6% of the men studied ( Figure   1).

Bone alterations in naïve patients.
Basal DXA was performed previous ART initiation. The median time elapsed between diagnosis and DXA scan was 3.3 month ( Table 1). As all the patients were under 50 years old Z-score was calculated, we found that 22,8% of the patients had a Z-score below -2 (Table 1). No signi cant differences were observed for the other risk factors studied.
In order to understand if any of the studied parameters affect Z-score at any location (LS or FN), a linear regression study was performed (Tables 2 and 3): We only observed a signi cant association of Z-Score in LS with CD8 and the CD4/CD8 ratio ( Table 2) and also with alcohol for FN measurement (Table 3). Likewise, when doing a logistic regression study of Z-score, assuming in this case normal and non-normal values, versus the same parameters under study; only the CD4/CD8 ratio appears to be associated in this case (Table 4). Therefore, this data leads us to think about the possible relationship between viral load and infection itself with the progressive bone deterioration of the HIV patient.

Discussion
How it can be extracted from the data obtained in our work, the results reveal a signi cant prevalence of bone involvement among newly diagnosed HIV-infected men before initiation of ART without any known secondary causes of osteoporosis, with 22.8% of the patients with a BMD lower than that expected for their age (Z-score <-2).
Additionally, a high percentage of these patients have low levels of vitamin D (87.6%).
Our data are similar to what has been previously described in other studies with patients of similar age groups. In this sense, Paccou J et al. (12) involving 49 naïve men, mean age was 31.6 ( 7.7) years demonstrated that the prevalence of low BMD was 24.5% 95% CI, 13.3-38.9 , similar to our ndings. In another study by Ceballos et al. (11) involving 70 naïve men, mean age 31 years , Low BMD (Z-score < -2.0) was found in 13% of the patients.
It is highly likely that the lifestyle of the study population, is an important factor behind such high rates of bone involvement. In our ndings we observed tobacco use (51.9%), no regular exercise (36.1%) and intermittent drug abuse (35.3%) as the most prevalent risk factors in our cohort. Previous studies have described that these factors may contribute to a decrease in BMD and an increased risk of fracture among patients infected with HIV. Nearly all these conditions have been found to be more prevalent among subjects with chronic HIV infection and ART experienced. (13)(14)(15)(16) Special attention should be given to vitamin D status and its impact on bone metabolism in these patients. In our cohort, 87.6% of the patients had low levels of vitamin D. In recent years, a number of studies have suggested that patients living with HIV infection have a high prevalence of vitamin D de cit independently of their geographic origin. (17)(18)(19) In addition to its deleterious impact on patients with HIV infection, vitamin D de ciency is a wellestablished risk factor for bone disease within the general population. (20) Indeed, recent publications suggest that the functions of vitamin D go beyond the skeleton, and that vitamin D may play a role in regulating cardiovascular and immunologic parameters.(21-23) Though some studies have described a protective role played by vitamin D in which this vitamin prevents loss of bone mass,(24) much remains unknown as to the degree to which vitamin D de ciency contributes to this loss and to an increase in risk of fracture among HIV-infected patients, so vitamin D should be included in the screening of bone fragility in this population (25) Currently, the primary guidelines and international consensus statements recommend that patients who are infected with the HIV virus undergo bone testing if they are over the age of 50 years (7,26,27) or with a history of pathologic fractures. These publications further advise clinicians to avoid ART regimens that pose a risk of bone toxicity, such as tenofovir disoproxil fumarate (TDF) and protease inhibitors, if the patient has existing bone involvement or fragility fracture. (1,7,16,28) Our ndings suggest that this recommendation may be revised, as over 22% of our study population, which consisted of MSM under age 50, had low BMD levels for their age. As there is still no curative treatment for HIV infection, it is foreseeable that these patients will continue requiring ART for years to come, thus putting them at an increased risk of loss of bone mass.
Among the limitations of the study, it should be noted that this is a single-center study may have in uenced the interpretation of some of our results, as a similar study performed in another geographic location may nd an increase or a decrease in the same parameters observed, mostly due differences in demographic, social and In short, the inclusion of DXA densitometry measurements and bone marker analysis as part of baseline evaluation of HIV-infected patients, although alone they would not be used to diagnose the disease in particular, but would provide clinical data to the physician to improve the health of the patient's bone mass, improve lifestyle habits that promote this bone comorbidity and avoid prescribing antiretroviral therapy that leads to bone loss. Similarly, a diagnosis of low BMD at an early age would affect the follow-up approach given to certain patients who by age do not undergo or consider densitometric parameters in the same way. As noted above, an increased risk of bone fracture has been found among patients with HIV infection compared to HIV-negative people; however, these studies have not found a correlation between abnormal DXA measurements and subsequent fracture. (31) As a result, we Consent for publication "Not applicable". Our manuscript does not contain data from any individual person.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
All data generated or analysed during this study are included in this published article.
Competing interests AC has received honoraria and speakers' fees from Gilead Sciences, MSD and ViiV. MG has received speakers' fees from ViiV.AM has led a patent on use of adenosine A2AR agonists to prevent prosthesis loosening (pending) and a separate patent on use of A2AR agonists and agents that increase adenosine levels to promote bone formation/regeneration. AM was supported by grants from "Instituto de Salud Carlos III" through the "Miguel Servet" Program (CP15/00053), co-funded by "Fondo Europeo de Desarrollo Regional (FEDER)". All authors have declared that no competing interests exist.