Several studies have associated non-nucleoside reverse transcriptase inhibitor (NNRTI) use with lower serum 25-hydroxy vitamin D [25(OH)D] levels [1–3]. Some investigators have attributed this effect to efavirenz (EFV) . The question of whether lower 25(OH)D levels represent an NNRTI class effect or can be related to a specific drug is of clinical importance, as the population at risk may merit more rigorous monitoring and supplementation. Furthermore, more data are needed to judge whether reported statistically significant differences are clinically relevant.
In response to the study by Pasquet et al.  and in order to evaluate the specific effects of EFV and nevirapine (NVP), we performed a secondary analysis of our longitudinal study published in a recent issue of AIDS . Furthermore, we tried to confirm our findings in an independent, cross-sectional study.
Both analyses were performed in the context of the Swiss HIV Cohort Study with patients' and Ethical committees' approval. In the longitudinal multicenter study, we measured 25(OH)D levels either during spring and fall season immediately before and 1 year after the initiation of combined antiretroviral treatment (cART). The actual analysis included 209 patients (75% men, 88% Caucasian, median age 37 years). Data from February to April (spring) and August to October (fall) were pooled after the exclusion of season-related differences in the NNRTI effects (data not shown).
The cross-sectional study was performed between March and May 2009 in Berne and included 262 unselected, consecutive individuals attending the HIV outpatient clinic (69% men, 79% Caucasian, median age 46 years). We compared 25(OH)D values in patients treated with EFV, NVP and boosted protease inhibitors (PI/rs) and calculated rates of 25(OH)D deficiency (<30 nmol/l) and insufficiency (<75 nmol/l). For the comparison of continuous variables we used t-tests (paired for the longitudinal study); for categorical variables χ2 tests. In the cross-sectional study, comparisons between treatment groups were adjusted for black ethnicity and IDU by linear regression, both of which have been associated with lower 25(OH)D values. All analyses were conducted with Stata 10 software (StataCorp LP, College Station, Texas, USA) using two-sided P values.
The longitudinal study provided 25(OH)D values before and 1 year after the initiation of cART (Fig. 1a). Whereas EFV was associated with a reduction of the median 25(OH)D from 46 to 34 nmol/l (P < 0.001), NVP and PI/r use correlated with small and nonsignificant increases. After 1 year of cART, 25(OH)D deficiency in EFV and NVP-treated patients occurred in 40.6 and 25.0%, respectively (P = 0.4). Consistently, EFV use was associated with an insignificantly higher proportion of 25(OH)D insufficiency (91.3 vs. 83.3%; P = 0.3).
An association of EFV with lower 25(OH)D levels was also seen in the cross-sectional study: compared with EFV-treated individuals, 25(OH)D levels were higher with NVP (P = 0.04) and PI/r (P = 0.048) (Fig. 1b). NVP-treated patients were more likely to have sufficient 25(OH)D levels than individuals exposed to EFV (17.9 vs. 5.4%; P = 0.05). Consistently, 25(OH)D deficiency was more prevalent in EFV than NVP-exposed individuals, yet without reaching statistical significance (41.3 vs. 32.1%; P = 0.5). Whereas treatment duration did not differ between groups (P = 0.7), there was an under-representation of black patients in the NVP as compared with the other groups (P = 0.05), and there were more IDU in the protease inhibitor as compared with the NNRTI groups (P = 0.005). Adjusting for these factors, 25(OH)D values remained significantly lower in the EFV than the protease inhibitor group (P = 0.006), but not in the EFV compared with the NVP group (P = 0.2).
In accordance with Welz et al. , 25(OH)D levels were consistently lower in EFV than in protease inhibitor-treated patients in both our studies. This difference could not be explained by an unequal distribution of black and IDU patients. Differences were small and unlikely to be clinically relevant. These findings are consistent with a case report that described severe 25(OH)D deficiency after starting EFV. EFV is thought to stimulate 25(OH)D catabolism through induction of cytochrome P450. Several enzymes of this family are involved in the anabolism (CYP27A1, CYP2R1, CYP3A4) and catabolism (CYP3A4 rather than CYP24) of 25(OH)D .
NVP use was not associated with lower 25(OH)D values in the longitudinal analysis, suggesting a specific effect of EFV rather than an NNRTI class phenomenon. This is in accordance with an earlier publication reporting no interaction between NVP and 25(OH)D . NVP also was associated with higher 25(OH)D values than EFV in the cross-sectional analysis. After adjusting for the unequal distribution of black ethnicity and IDU among the subgroups, however, this difference lost statistical significance, which at least partially is explained by the small number of NVP-treated patients.
The somewhat higher 25(OH)D values in the longitudinal analysis are explained by the fact that 50% (vs. 0% in the cross-sectional study) of the measurements were performed in fall after peak sun exposure.
In our analyses, EFV treatment was associated with lower 25(OH)D levels compared with both protease inhibitors and NVP. Part of the difference between the NNRTIs was due to important confounders that must rigorously be controlled for. Still, our data rather support the hypothesis of an EFV-specific effect. Considering the relevant proportion of patients with 25(OH) deficiency in all treatment groups and the postulated benefits of vitamin D, we believe that screening and correction of 25(OH)D deficiency should not be restricted to EFV-treated individuals but offered to all patients. Specific CYP polymorphisms and classical risk factors, such as black ethnicity and IDU, may define patients in whom EFV will result in clinically relevant 25(OH)D deficiency .
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