The role of vitamin D status on treatment outcome among HIV- infected children receiving care in Kisumu County, Kenya

Abstract Background: Kenya has a paediatric HIV burden of nearly 140,000 children, of which only 48% of those on antiretroviral therapy (ART) have achieved the desired viral suppression possibly due to vitamin D deficiency. We explored the influence of vitamin D levels on treatment outcome. Method: We performed a cross-sectional study of 196 participants aged 3 – 14 years; among them 98 HIV infected who received treatment between 2019 - 2020 in Jaramogi Oginga Odinga Teaching and Referral Hospital, Kenya. The exposure was vitamin D levels, including deficient (<20 ng/ml), insufficient (≥20 - <30 ng/ml), and sufficient (30 – 50ng/ml). The outcome was optimal immune recovery (CD4 ≥ 500 cells/mm3) and optimal viral suppression (viral load ≤ 200 copies/ml). We compared difference in means for each vitamin D category between HIV infected and uninfected using independent t-test, multiple comparisons of vitamin D levels among age categories using ANOVA and post hoc test and Pearson correlation to correlate vitamin D levels, CD4 and viral load of HIV infected children. Results: Compared with HIV uninfected, HIV infected recorded mean age ± standard deviation of10.65±2.17 years with 39(39.8%) males vs. 6.68±2.81 years with 52(53.1%) males p<0.001; and the difference in vitamin D mean levels was statistically significant [28.21 ± 6.39 infected vs.30.88 ± 6.62 uninfected] t = 2.94, df =194, p = 0.004, 95%CI (0.90 – 4.59). Among age categories, mean vitamin D varied significantly F (2,193) = 10.68, p =0.001; with higher levels observed between 1-4 years category {mean difference 4.64ng/ml, p = 0.02, [95%CI 1.49 – 7.78]} and 5-9 years category {mean difference 4.33ng/ml, p = 0.001, [95%CI 1.89 – 6.38]} as compared to 10 – 14 years respectively. Additionally, children with optimal immune recovery recorded higher proportion of vitamin D deficiency and insufficiency (12.24% and 42.86%) as compared with sub optimally recovered 1.02% and 4.08%); while children with optimal viral suppression recorded higher proportion of vitamin D deficiency and insufficiency (8.16% and 30.61%) as compared with sub optimally suppressed (5.1% and 16.3%). Conclusion : Infections with HIV suppresses levels of vitamin D, but this has no influence on CD4 counts and viral load status in children receiving ART.


Introduction
About 40% of the 1 million HIV infected children < 15 years of age, receiving antiretroviral treatment (ART) care in sub Saharan Africa have not achieved the desired viral suppression (de ned as viral load < 200 copies /ml) despite improved implementation of more e cacious 1st line ART regimens [1], possibly due to vitamin D de ciency.Plasma vitamin D [de ned as 25 hydroxyvitamin D (25(OH)D] is a steroid hormone, fat-soluble micronutrient obtained primarily from ultraviolent B rays and plays crucial roles of balancing homeostasis, regulating mineral metabolism, bone maintenance, cell growth, and immune response useful in containing infectious diseases [2][3][4][5].De ciency of vitamin D therefore, promotes in ammatory responses by upregulating in ammatory markers such as IL-6 and TNF-α overproduction in HIV-infected patients [2,6], leading to renal 1-hydroxylase impairment with reduction of parathyroid hormone (PTH) stimulatory effect on the production of hormonally active 1,25 dihydroxyvitamin D [1,25(OH)2D] [7].Additionally, vitamin D de ciency accelerates faster progression of HIV disease and perfectly correlates with decreased patient survival [3,8].Thus, there is a need for periodic evaluation of levels with subsequent supplementation where necessary during infections and treatment.
Conversely, the body immune system such as the brain, heart, stomach, lymphatic system, skin, and prostrate tissue are composed of CD4 + T-cells and B-cells that express vitamin D receptors (VDR) useful in improving immune functions and reduction of in ammations [9,10].Interactions between VDR and Vitamin D mediate anti-in ammatory effects on both innate and adaptive immune systems that regulate immunity in the context of bacterial or viral in ammation [10].Consequently, severe vitamin D de ciency is linked to low CD4 + cell counts and increased markers of in ammation in HIV-infected patients [5,11] suggesting a possible relationship among vitamin D, infectious diseases and immune functions in pediatric population that highlights the role and need for supplementation in cases of de ciencies [10,12].
Moreover, studies have also shown that HIV infected participants on ART, and who received vitamin D supplementation to su cient levels recorded much improved viral suppression compared to their counterparts who experienced low levels of vitamin D [13]; thus, suggesting a possible in uence of vitamin D on treatment outcome.Kenya has nearly 140,000 children living with HIV (CLHIV) < 15 years of age and 8000 new pediatric infections annually, but only 48% of those on ART have achieved the desired viral suppression [14].In Kisumu County, adolescents and young adults living with HIV (AYALH) has continued to register suboptimal viral suppression of 84.4% overtime despite increasing availability of improved services [15], and vitamin D de ciency has been suggested as a potential risk factor driving this poor outcome [16].

Method
Using Sharma 2019 formula, blood samples of 196 [98 HIV infected] consenting participants aged 3-14 years from Kisumu County was determined for a cross-sectional study by systematic sampling.
Laboratory tests were conducted at Jaramogi Oginga Odinga teaching and referral hospital (JOOTRH), Kenya from November 2019 to March 2020 based on large volume of participants receiving comprehensive care, and quality standards available.Permission to conduct study was obtained from JOOTRH ethical review committee, and participants were enrolled upon receipt of guardian informed written consent.
Children on vitamin D supplements were excluded from the study.Demographic details were extracted from clinic records by nurse counselor who equally collected 2ml of venous blood sample into each plain and EDTA vacutainer tubes (BD Franklin Lakes, USA) for evaluation of viral load, vitamin D and CD4 + cells levels, respectively.Sera were tested for vitamin D using Biomerieux® Mini Vidas automated immunoassay analyzer, and HIV-1 RNA using Roche diagnostics® COBAS real time PCR test, while, CD4 + cell count was performed using Becton Dickinson® uorescence-activated cell sorter (FACS) system.All tests were done according to manufacturers' instructions.Statistical analysis was performed using SPSS version 20.
We compared difference in means for each vitamin D category between HIV infected and uninfected using independent t-test, multiple comparisons of vitamin D levels among age categories using ANOVA and post hoc test and Pearson correlation to correlate vitamin D levels, CD4 and viral load of HIV infected children.

Results
Overall, we recruited 196 participants of which 98 were HIV infected age range 5-14 years with another 98 HIV uninfected (control group) age 3-14 years.HIV infected participants recorded a mean age ± standard deviation of 10.7 ± 2.17 years with a median (IQR) duration of ART of 6.2 years (3.1-13.2),39.8% being male, and a mean vitamin D level of 28.21 ± 6.39; as compared to uninfected group having a mean age ± standard deviation of 6.7 ± 2.81, 53.1% being males and a mean vitamin D level of 30.88 ± 6.62.Overall, the difference in mean vitamin D between HIV infected and uninfected participants was statistically signi cant [t = 2.94, df 194, p = 0.004 (Table 1).However, using vitamin D de ciency, insu ciency and su ciency cut-off values of < 20 ng/ml; ≥20 -<30 ng/ml; and 30-50ng/ml respectively, HIV infected participants recorded mean de ciency, insu ciency and su ciency levels of 18.1ng/ml, 25.9ng/ml, and 34.3ng/ml as compared to 18.4ng/ml, 25.5ng/ml and 35.7ng/ml respectively, for the uninfected group with no statistical differences.

Discussion
Although 25(OH) D levels are commonly used to de ne vitamin D status, its suitability and applicability as a predictor for good prognosis in children on HIV care remains debatable.In our study, we examined the relationship between age categories and levels of vitamin D. Our results revealed that levels of vitamin D in children increases with increasing age during1-4 years to reach peak levels of 31.91ng/ml and begin to decline (Fig. 1).Supposedly, this value represents the physiologically ideal level of vitamin D much required to optimize various vitamin D high level viremia-associated health outcomes and physiological parameters such as bone mineral density [17]; and is perfectly comparable with 36.5ng/mlpeak levels at 18 month postnatal earlier reported in a Malawi study [18], Furthermore, our nding is supported by Mogire et al [19] and Alvarez-Rodriguez et al [20] who equally observed a decreasing trend with vitamin D levels as age increases; and associated the same with a change in expression and defective function of some toll-like receptors (TLRs) involved in viral response [20].To the contrary, a similar study among HIV uninfected infants in our neighborhood Tanzania observed that low levels of vitamin D was common in infants during their early infancy, especially those under exclusive breastfeeding.[21], the cause of which remain unclear.
Furthermore, we compared the mean levels of vitamin D between HIV infected and uninfected children receiving antiretroviral therapy (ART) with those on routine outpatient visits.Our data analysis revealed that HIV infected children profoundly experienced low levels of vitamin D as compared to their uninfected counterparts.Interestingly, this is explained by the fact that persons living with HIV (PLWH), and receiving ART particularly protease inhibitor (PIs) or non-nucleoside reverse transcriptase inhibitors (NNRTIs) traditionally experience suppressed levels of vitamin D in blood [22], but which becomes more exacerbated in individuals receiving efavirenz (EFV) [5,23]; the mechanism of which is under investigation.Moreover, much lower levels of serum vitamin D have equally been observed among late ART-initiated children (18 months to 12 years) as compared to those on early-ART initiation in the rst year of life [22], but this is explained by the in uence of increasing age on vitamin D levels reported in our study; and is consistent with children physiological parameters such as bone mineral density [17].Thus, our nding supported an earlier systematic review and meta-analysis report showing increased risk of vitamin D de ciency in PLWH compared to uninfected subjects; more so to participants receiving ART, and in older age category [24].Slightly lower levels of vitamin D among HIV infected infants and adults on ART have equally been reported in a similar study in Botswana [4], although this was partly attributed to demographic and dietary habits of consuming more of traditional and indigenous foods enriched with better dietary diversity [4] Additionally, we compared the proportion of Immunocompromised participants with Immunocompetent group based on their vitamin D levels, and established that majority participants who experienced vitamin D de ciency or insu ciency were actually Immunocompetent rather than Immunocompromised; and consistent with earlier ndings by Kakalia et al, [25].To the contrary, our ndings dispelled earlier report suggesting a possible correlation between vitamin D levels and CD4 + T-cell counts [3,5,26].Instead, vitamin D concentrations above 30 ng/ml has been found to be more associated with mortality of HIV infected children compared to levels lower than 10 ng/ml [21].Meanwhile, a good number of local and international studies have reported varying relationship; both positive and negative, with others failing to demonstrate any correlation [27] A recent study in the neighborhood Uganda associated low CD4 count with vitamin D de ciency [5]; thus, echoing other ndings from related studies in France and Colombia which equally suggested that severe vitamin D de ciency could be independently associated with low CD4 count [3,4,28].Although high CD4 cells counts have been associated with higher vitamin D levels [29], while low vitamin D levels is linked to immune dysfunction that also in uence the expression of in ammatory markers [24], we could not establish this outcome owing to the scope of our study.Moreover, it is good to note that children in our study had been on ART for quite some times, majority of whom were experiencing good health status by the time of sampling; hence, the immunologic, HIV stage and other HIV related factors may have normalized and compensated for the effects of vitamin D status.We however still believe that this could have been a bit different in children who may have had advanced disease at ART initiation.
Moreover, we compared the proportions of participants who achieved optimal viral suppression visa-vi those with sub optimal suppression and their levels of vitamin D; and found that those who were optimally suppressed were in fact the majority with either vitamin D de ciency or insu ciency contrary to earlier suggestion by Stalling et al that vitamin D supplementation intervention to su cient levels helps to improve viral suppression of patients on ART [13] Our ndings equally dispute earlier suggestion by Overton et al that high dose supplementation of vitamin D with ART substantially enhance viral suppression [30].
This study included a number of limitations.First, the small sample size and single site experience limits generalizability and requires further study in multisite samples.Additionally, our study was performed entirely in Kisumu western Kenya, an equatorial region abundant with sunshine which may have compensated for de ciency attributed to diet or HIV infection; thus, generalizability to other regions with less sunshine may be limited.Finally, our population consisted of children with relatively preserved immune function, having been on ART for sometimes and therefore our failure to accurately demonstrate the relationship between vitamin D status, CD4 count and viral load; hence, cannot be generalized to children with more advanced HIV disease and low baseline CD4 counts.

Conclusion
In early childhood, vitamin D increases with increasing age to optimal levels by age 5-9 years during which it stabilizes and begin to decrease with advancing age.Our data indicate that infections with HIV disproportionately in uence levels of vitamin D in children, although this has no effect on CD4 counts and viral load.We recorded a low prevalence of vitamin D de ciency and insu ciency in a small cohort, and suggest that a further study with ART naïve participants with larger sample size would be ideal to better understand possible confounding variables that may affect vitamin D status in HIV infected children MSA and LO conceived and designed the study with inputs from CGO; and performed statistical analysis with additional input from BG.All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of data analysis.MSA and CGO contributed to acquisition, analysis, and interpretation of data; and drafted the manuscript with critical revision from LO and BG.All authors read and approved the nal manuscript.

Funding
Research reported in this publication was supported in part by Fogarty International Center of the National Institute of Health (NIH) under Award Number D43TW011306.The content is solely the responsibility of the authors and does not necessarily represent the o cial views of the National Institute of Mental Health (NIMH).The NIMH had no role in the design and conduct of the study; collection, management, analysis, interpretation of data, review or approval of the manuscript; and decision to submit the manuscript for publication. Figures

Table 2 Multiple
Comparisons of vitamin D levels among age categories based on ANOVA and post hoc test *.The mean difference is signi cant at 0.05 level.