Timing, Dosage, and Adherence of Antiretroviral Therapy and Risk of Osteoporosis in Patients With Human Immunodeficiency Virus Infection in Taiwan: A Nested Case-Control Study

The progression of acquired immunodeficiency syndrome is delayed in patients with human immunodeficiency virus (HIV) infection receiving antiretroviral therapy (ART). However, long-term ART is associated with adverse effects. Osteoporosis is one of the adverse effects and is a multifactorial systemic skeletal disease associated with bone fragility and an increased risk of fracture. We performed a longitudinal, comprehensive, nested case-control study to explore the effect of ART on the risk of osteoporosis in 104 osteoporotic and 416 non-osteoporotic patients with HIV infection at their average age about 29 years old in Taiwan. Patients with history of ART, current exposure to ART, higher cumulative defined daily doses (DDDs), or higher ART adherence were at a higher risk of osteoporosis (p < 0.05). Patients receiving nucleoside/nucleotide reverse transcriptase inhibitor (NRTI)-containing regimen (zidovudine-lamivudine combination, lamivudine-abacavir combination, and abacavir alone) and protease inhibitor (PI)-containing regimen (lopinavir-ritonavir combination, ritonavir, and atazanavir) had a higher risk of osteoporosis (p < 0.05). Especially, patients receiving high doses of the PIs lopinavir-ritonavir combination had an increased risk of osteoporosis (p < 0.05). In conclusion, history of ART, current exposure to ART, higher cumulative DDDs, and higher ART adherence were associated with an increased risk of osteoporosis. Furthermore, NRTI- and PI-containing regimens and high doses of PIs lopinavir-ritonavir combination may be associated with an increased risk of osteoporosis in patients with HIV infection in Taiwan.

Among these, approximately 23.3 million were receiving antiretroviral therapy (ART). After ART, HIV replication is effectively inhibited and HIV-infected immune cells become latent (Pham and Mesplede, 2018;Zhang et al., 2019). Patients with HIV/AIDS who receive ART demonstrate delayed AIDS disease progression, improved quality of life, and lower all-cause mortality (Antiretroviral Therapy Cohort, 2017;Lu et al., 2018). These patients need to receive ART regularly on a lifelong basis.
Osteoporosis is a multifactorial systemic skeletal disease accompanied by low bone mineral density, deterioration of bone architecture, bone fragility, and consequently increased fracture risk (Ji and Yu, 2015;Sozen et al., 2017;Liu et al., 2019). Loss of bone mineral density is frequently observed in patients with HIV infection receiving ART (Duvivier et al., 2009;Van Vonderen et al., 2009;Grant et al., 2016;Hoy et al., 2017). Moreover, patients with HIV infection receiving PI-containing regimens demonstrate bone loss in the spine, whereas those receiving NRTI-containing regimens show bone loss in the hip (Hoy et al., 2017). Other studies have reported that a combination of NRTI drugs -and PI-containing regimens also lead to bone loss among patients with HIV infection (Duvivier et al., 2009;van Vonderen et al., 2009).
Previous studies have reported that patients with HIV infection receiving ART are at a higher risk of bone loss (Duvivier et al., 2009;Van Vonderen et al., 2009;Grant et al., 2016;Hoy et al., 2017). However, the correlation of ART usage, timing, dosages, and adherence with osteoporosis risk among patients with HIV infection remains unclear. In this longitudinal nested case-control study, a comprehensive database was used to explore the association between ART and the risk of osteoporosis among patients with HIV infection in Taiwan. Detailed associations between usage, timing, dosages, and adherence to ART and the risk of osteoporosis were investigated.

Study Cohort
This nested case-control study was approved by the Human Studies Committee of China Medical University Hospital, Taichung, Taiwan (approval number: CMUH107-REC3-074). We identified 14,165 patients with HIV infection (International Classification of Diseases, Ninth Revision, Clinical Modification [ICD-9-CM]: codes 042, 043, 044, and V08) aged under 35 years whose data were recorded in the National Health Insurance Research database (http://nhird. nhri.org.tw/) between 1998 and 2011 ( Figure 1).
A total of 11,126 patients were included in the study after applying the following exclusion criteria: patients with a diagnosis of osteoporosis before human immunodeficiency virus (HIV) infection (N 177), patients with antiretroviral therapy (ART) prescriptions before HIV infection (N 10), patients with a diagnosis of pathologic or open fracture during the study period (N 2,572), or patients with cancers during the study period (N 280).

Cases and Controls
As cases, we included patients who had reported osteoporosis for the first time after HIV infection during the study period (N 105) ( Figure 1 and Table 1). The date of osteoporosis diagnosis was defined as the index date. The controls were patients with HIV infection who did not develop osteoporosis during the study period (N 11,021) ( Figure 1 and Table 1). To prevent potential bias, the density sampling matching method (1:4 ratio for cases and controls) was applied to match osteoporosis and nonosteoporosis groups based on age, sex, and the first date of HIV infection diagnosis. After matching, 104 osteoporosis cases and 416 non-osteoporosis controls were included in the study ( Figure 1 and Table 1).

Exposure to ART Drugs
Exposure was defined as the usage of ART drugs during the study period ( Table 2) Table S1). The currently used ART drugs and also available in our database were shown ( Figure 2; Supplementary Table S2).
For determining the duration of exposure to ART drugs, we further categorized exposure into non-exposure, current exposure (0 < YEAR ≤ 1), recent exposure (1 < YEAR ≤ 2), and past exposure (2 < YEAR) groups based on the "latest" ART prescription date prior to the index date (Table 2 and Figure 3). The index date was defined as the diagnosed date of osteoporosis. For the current exposure patients, patients were defined if they had ART drug availability during the time window of 0-1 year before the index date ( Figure 3). For the recent exposure patients, patients were defined if they had ART drug availability during the time window of 1-2 years before the index date ( Figure 3). For the past exposure patients, patients were defined if they had ART drug availability during the time window of more than 2 years before the index date ( Figure 3). Current exposure patients included the patients who continuously used ART drugs since HIV infection (Figure 3). For the cumulative defined daily dose (DDD), we further categorized cumulative DDD (usage dose) into non-use, cumulative DDDs < 2500, and cumulative DDDs ≥ 2500 during the study period. For adherence to ART, we further classified the usage adherence into non-exposure, low adherence (0 < adherence (ADH) ≤0.8), and high adherence (0.8 < ADH) during the study period.

Patient Data Collection
The data (the National Health Insurance Research database) included longitudinal inpatient and outpatient records of age, sex, admission, prescription, diagnosis, procedures, and ambulatory care. In this study, demographic data (age, sex, follow-up period, comorbidities, and ART usage) were collected for patients with HIV infection ( Table 1)  Cumulative DDD and adherence were calculated during the study period. Cumulative DDD was defined as the total dose of ART administered to a patient from the initiation of ART treatment to the end of study. Adherence was defined as follows: (total number of prescribed days from ART initiation to study end)/(total number of observation days for a patient from ART initiation to study end).

Statistical and Subgroup Analyses
Categorical variables, including sex, comorbidities, and ART usage, were analyzed using the Chi-square test between osteoporosis cases and non-osteoporosis controls ( Table 1). Continuous variables, including age and follow-up period, were analyzed using Student's t-test (Table 1). Conditional logistic regression analysis was applied to investigate the association between ART use and osteoporosis risk in patients with HIV infection (Table 2; Figure 2; Figure 4). Subgroup analyses according to NRTI-, PI-, NNRTI-, other ART-containing, and over two ART drugs-containing regimens were used to investigate the association between    ART use and osteoporosis risk during the study period ( Figure 2). Subgroup analyses for specific NRTI-and PIcontaining regimens with dose-response relationships were performed to explore the association between the dose effect of specific NRTI-and PI-containing regimens and osteoporosis risk (Figure 4). Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Results with p-values < 0.05 were considered significant. Statistical analyses were

Basic Characteristics of Study Subjects
The study subjects comprised 105 patients with osteoporosis and 11,021 non-osteoporosis controls ( Figure 1; Table 1). Significant differences were observed regarding age, sex, follow-up period, and comorbidities between the two groups (p < 0.05). Patients with osteoporosis included more women, were older, had shorter follow-up years, and had increased incidence of comorbidities (rheumatic disease and diabetes with or without chronic complications). Furthermore, the matched osteoporosis cases included a higher number of cases of chronic pulmonary disease, rheumatic disease, and diabetes with or without chronic complications ( Table 1). The exposure to antiretroviral therapy (ART) was defined as the use of ART drugs during the study period (Figures 1, 3).

ART Usage, Timing, Dosage, and Adherence and Osteoporosis Risk in Patients With HIV Infection
Conditional logistic regression analysis was used to investigate the association between ART usage and osteoporosis risk adjusted for age and comorbidities ( Table 2). Table 2, there were significant differences in ART usage, duration, dosage, and adherence (p < 0.05). The patients who received ART had a higher risk of osteoporosis, with an odds ratio (OR) of 2.11 (95% confidence interval (CI): 1.22-3.66) than those who did not use ART during the study period (p 0.0077; Table 2). Patients with current exposure to ART had a higher risk of osteoporosis, with an OR of 2.51 (95% CI: 1.38-4.56) than those without exposure to ART (p 0.0026) ( Table 2). However, the associations were not statistically significant among patients with recent or past exposures (p > 0.05) ( Table 2).
These results suggest that patients were at higher risk of osteoporosis when they received ART, were currently exposed, had higher cumulative DDDs, or had higher ART adherence.

NRTI-, PI-, and NNRTI-Containing Regimens and Risk of Osteoporosis in Patients With HIV Infection
To investigate which types of ART-containing regimens were associated with increased risk of osteoporosis, patients receiving nucleoside/nucleotide reverse transcriptase inhibitor (NRTI)-, protease inhibitor (PI)-, non-nucleoside reverse transcriptase inhibitor (NNRTI)-, other ARTcontaining, and over two ART drugs-containing regimens were investigated (Supplementary Table S1). The currently used ART drugs and available in our database were shown ( Figure 2). For the NRTI-containing regimen, abacavir were associated with higher risks of osteoporosis in patients receiving ART than in those not receiving ART (p < 0.05) (Figure 2). Patients receiving PI-based ART had a higher risk of osteoporosis, with an OR of 2.78 (95% CI: 1.52-5.10), than those not receiving ART (p < 0.001) (Figure 2). Among the PI drugs, ritonavir and atazanavir were associated with higher risks of osteoporosis in patients receiving ART than in those not receiving ART (p < 0.001) (Figure 2). There were no sufficient numbers in darunavir (PI)-and raltegravir (integrase strand transfer inhibitor (INSTI))-containing regimens. The association was not statistically significant among patients receiving NNRTIs (p > 0.05) (Figure 2). For over two ART drugs-containing regimens, lamivudine and abacavir (NRTI/NRTI), lopinavir and ritonavir (PI/PI), and zidovudine and lamivudine (NRTI/NRTI) were associated with higher risks of osteoporosis in patients receiving ART than in those not receiving ART (p < 0.05) (Figure 2).
These results suggest that patients were at an increased risk of osteoporosis when they received NRTI-and PI-containing regimens. Patients receiving the NRTIs zidovudine-lamivudine combination, lamivudine-abacavir combination, or abacavir were at a risk of osteoporosis. Patients receiving the PIs lopinavirritonavir combination, ritonavir, and atazanavir were at an elevated risk of osteoporosis.

Dose-Response Relation of NRTI-Containing and PI-Containing Regimens and Risk of Osteoporosis in Patients With HIV Infection
Three NRTIs, zidovudine-lamivudine combination, lamivudineabacavir combination, and abacavir, were associated with an increased risk of osteoporosis (p < 0.05) (Figure 2). However, patients receiving higher doses of these drugs had no significantly increased risk of osteoporosis compared with those not receiving ART (Figure 4). Three PIs lopinavir-ritonavir combination, ritonavir, and atazanavir were associated with an increased risk of osteoporosis (p < 0.05) (Figure 2). Patients receiving higher doses of lopinavir-ritonavir combination were at a significantly higher risk of osteoporosis than those not receiving ART (Figure 4).
These results suggest that patients were at an increased risk of osteoporosis when they had higher doses of the PI-containing regimen lopinavir-ritonavir. However, patients with higher doses of NRTIs had no significantly increased risk of osteoporosis.

DISCUSSION
In this nested case-control study, we investigated the association between antiretroviral therapy (ART) and the risk of osteoporosis in patients with human immunodeficiency virus (HIV) infection in Taiwan. We found that ART usage, current exposure, higher cumulative defined daily doses (DDDs), and higher ART adherence were associated with an increased risk of osteoporosis. Furthermore, nucleoside/nucleotide reverse transcriptase inhibitor (NRTI)-and protease inhibitor (PI)containing regimens and higher doses of PIs-lopinavirritonavir combination-may be associated with an increased risk of osteoporosis in patients with HIV infection in Taiwan. Therefore, this study showed that ART, especially NRTIcontaining and PI-containing regimens, may be potential risk factors for osteoporosis. Switching from ART regimens may be one option to improve bone health in patients with HIV infection (Ciccullo et al., 2018).
In our study, patients with HIV infection were at a higher risk of osteoporosis at a relatively young age. ART usage, current exposure, higher cumulative DDDs, and higher ART adherence were associated with osteoporosis risk. Osteoporosis, characterized by low bone mineral density and deterioration of bone architecture, is usually associated with old age and is more prevalent in women (Kanis et al., 2008). The current exposure patients were those who had ART drug availability during the time window of 0-1 year before osteoporosis. Current exposure patients included the patients who continuously used ART drugs since HIV infection. Why current exposure to ART were associated with higher risk of osteoporosis may be due to patients may have higher cumulative DDDs since HIV infection. Bone mineral density screening has been suggested for male patients with HIV infection older than 50 years and in postmenopausal women (Aberg et al., 2014;Gonciulea et al., 2017). In agreement with our results, previous study findings reveal that bone loss is observed in patients with HIV infection who receive ART (Mccomsey et al., 2008;Duvivier et al., 2009;Van Vonderen et al., 2009;Haskelberg et al., 2012;Grant et al., 2016;Hoy et al., 2017;Komatsu et al., 2018).
Our results demonstrated that patients receiving NRTIcontaining regimen had a higher risk of osteoporosis. Studies have reported that NRTI-containing regimens, including zidovudine-lamivudine combination therapy and tenofovir monotherapies, induce osteoporosis or osteoporotic fractures (Van Vonderen et al., 2009;Haskelberg et al., 2012;Grant et al., 2016;Komatsu et al., 2018). Furthermore, our study showed that patients receiving zidovudine-lamivudine combination, lamivudine-abacavir combination, and abacavir had a higher risk of osteoporosis. In agreement with our results, van Vonderen et al. reported that patients with HIV infection treated with zidovudine and lamivudine combination had greater bone loss (Van Vonderen et al., 2009). Zidovudine and lamivudine stimulate osteoclastogenesis in vitro and cause osteopenia in mice by increasing the promoter activity of tartrateresistant acid phosphatase and the binding of nuclear factorkappa B (Pan et al., 2004;Pan et al., 2006).
Our results showed that patients receiving PI-containing regimen had a higher risk of osteoporosis. Among the PIs, lopinavir-ritonavir combination, ritonavir, and atazanavir were associated with a higher risk of osteoporosis. Furthermore, our results suggested that high doses of three PIs lopinavir-ritonavir combination were associated with an increased risk of osteoporosis. In agreement with our results, previous findings revealed lower bone mineral density in patients treated with ritonavir (Dimeglio et al., 2013). Increased osteoclast activity has been observed in the presence of ritonavir (Jain and Lenhard, 2002). Reportedly, ritonavir enhances osteoclast differentiation via upregulation of the production of osteoclast growth factors (Modarresi et al., 2009).
The limitations of this study include not considering factors such as genetic background information (both virus genotype and human genetics); environmental factors (nutrition level, education, job stress, and exercise); and other clinical characteristics, including body mass index, bone density, blood HIV viral load, blood cluster of differentiation antigen 4 (CD4) count, bone mineral density test results (for example: dual energy x ray absorptiometry (DXA)-based examinations), and serum bone turnover markers. Another limitations of this study include the lack of information on current therapies, such as integrase inhibitors or tenofovir alafenamide (TAF)-based therapies (Supplementary Table S1).
In conclusion, this was a longitudinal and broad study that elucidated the association of ART with the risk of osteoporosis using a database of patients with HIV infection in Taiwan. Patients receiving NRTI-or PI-containing regimens have a higher risk of osteoporosis. These patients are suggested to have regular examinations for bone mineral density when they are administrated with NRTI-or PI-containing regimens. This information may help identify therapeutic options regarding long-term ART for the prevention of adverse effects, particularly osteoporosis, in patients with HIV infection.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the nested case-control study under Human Studies Committee of China Medical University Hospital, Taichung, Taiwan ( approval number: CMUH107-REC3-074). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.