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Low maternal vitamin D is associated with increased risk of congenital and peri/postnatal transmission of Cytomegalovirus in women with HIV

Low maternal vitamin D is associated with increased risk of congenital and peri/postnatal... transmission of Cytomegalovirus in women with HIV. PLoS ONE 15(2): e0228900. https://doi.org/ 10.1371/journal.pone.0228900 Background Editor: Michael Nevels, University of St Andrews, CMV infection of the fetus or neonate can lead to devastating disease, and there are no UNITED KINGDOM effective prevention strategies to date. Vitamin D is a potent immunomodulator, supports Received: October 15, 2019 antiviral immune responses, and plays an important role in placental immunity. Accepted: January 24, 2020 Published: February 13, 2020 Methods Peer Review History: PLOS recognizes the Retrospective cohort study to evaluate the impact of low maternal vitamin D on congenital benefits of transparency in the peer review and early postnatal transmission of CMV among HIV-infected, non-breastfeeding women process; therefore, we enable the publication of and their HIV exposed but negative infants from an urban HIV clinic. Vitamin D panel was all of the content of peer review and author responses alongside final, published articles. The performed on stored maternal plasma obtained near time of delivery. Infant CMV testing at editorial history of this article is available here: 0–6 months included urine and oral cultures, and/or serum polymerase chain reaction https://doi.org/10.1371/journal.pone.0228900 testing. Copyright:© 2020 Bearden et al. This is an open access article distributed under the terms of the Results Creative Commons Attribution License, which permits unrestricted use, distribution, and Cohort included 340 mother-infant pairs (births 1991–2014). Among 38 infants (11%) with a reproduction in any medium, provided the original CMV+ test between 0–6 months, 4.7% (14/300) had congenital CMV transmission (CMV+ author and source are credited. test 0–3 weeks), and 7.6% (24/315) had peri/postnatal CMV (CMV+ test>3 weeks-6 Data Availability Statement: We are unable to months). Women with lower calcitriol (1,25-dihydroxyvitamin D), the active form of vitamin publicly share the dataset from this study due to ethical and legal restrictions. According the D, were more likely to have an infant with congenital (OR 12.2 [95% CI 1.61–92.2] P = 0.02) PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 1 / 15 Low vitamin D linked to congenital CMV University of Southern California’s Office of Ethics and peri/postnatal (OR 9.84 [95% CI 2.63–36.8] P = 0.0007) infections in multivariate analy- and Compliance, there is no way to make the ses, independent of maternal HIV viral load and CD4 count. dataset truly de-identified. The dataset is made up solely of women with a diagnosis of HIV and their offspring. The diagnosis of HIV is an indirect Conclusion identifier and considered sensitive information requiring additional protections. Additional indirect This study demonstrates an association between inadequate maternal calcitriol during preg- identifiers in the dataset include age, race/ethnicity, nancy and increased congenital and early postnatal acquisition of CMV among non-breast- and sex. Additionally, the consent signed by the feeding women with HIV and their HIV negative infants. study participants did not include a provision for their data to be made publicly accessible. Data requests can be directed to department administrator, Carlota Obnillas, obnillas@usc.edu, and if the request is approved, the USC Stevens Center for Innovation will assist and create a Data Transfer Agreement. Introduction Funding: This work was supported by the SC CTSI Human Cytomegalovirus (CMV) infection is the most common congenital infection world- (https://sc-ctsi.org), (NIH/NCRR/NCATS) through wide. It is the top non-genetic cause of childhood deafness in the world and can lead to neuro- Grant UL1TR000130, awarded to AK and AB. Its logic and neurodevelopmental disorders, multisystem illness, growth and development contents are solely the responsibility of the authors abnormalities, and death.[1,2] Approximately 50–70% of women of childbearing age in devel- and do not necessarily represent the official views oped countries are CMV infected, with the highest prevalence among women of lower socio- of the National Institutes of Health. This study was funded in part by a seed grant provided by the economic status.[3] Seroprevalence approaches 100% among women of child-bearing age in Department of Obstetrics and Gynecology at the resource-limited countries and in those with Human Immunodeficiency Virus (HIV) infec- University of Southern California awarded to KV. tion.[2,4] Mother-to-child-transmission (MTCT) of CMV can occur prenatally (congenital RP received support in the form of a salary from infection), during birth, and postnatally through breast milk.[5] Mothers and other caregivers Pan Laboratories, Irvine, CA. The funders had no can also transmit CMV to their infants postnatally through infected secretions.[2] Maternal role in study design, data collection and analysis, decision to publish, or preparation of the CMV infections and reactivations are often asymptomatic and unnoticed, and unlike HIV, manuscript. The specific roles of these authors are there are currently no effective strategies widely implemented for the prevention of MTCT of articulated in the ‘author contributions’ section. CMV.[6] Rates of congenital CMV are often higher among infants of women with HIV infec- Competing interests: RP is the salaried Director of tion, making them an ideal population for study.[2,4,7–11] Pan Laboratories, Irvine, CA. There are no patents, Vitamin D is obtained either from exposure to ultraviolet light or from the diet. In addition products in development or marketed products to to its role in calcium homeostasis and skeletal health, Vitamin D is a well-known and potent declare. This does not alter our adherence to PLOS modulator of the immune system.[12] Vitamin D supports immune system antiviral responses ONE policies on sharing data and materials. through the induction of autophagy and production of antimicrobial peptides like cathelicidin, and likely plays an important role in helping to protect the developing fetus from infections during pregnancy.[13–16] A multitude of cells in the body have the vitamin D receptor and many cells, including the cells of the placenta, also have the ability to convert 25-hydroxyvita- min D (25(OH)D), the main circulating form of vitamin D, to its bioactive form, 1,25-dihy- droxyvitamin D (1,25(OH)D ).[17,18] This allows for local production of 1,25(OH)D and the 2 2 subsequent vitamin D-dependent antimicrobial immune responses in the setting of specific conditions or stimuli.[15,18–20] Vitamin D’s important role in supporting the immune system’s antiviral functions, includ- ing those at the level of the placenta, suggests its relevance to MTCT of CMV in utero. Addi- tionally, vitamin D may contribute to the immune system’s ability to limit viral shedding and therefore play a role in limiting perinatal and early postnatal CMV transmission. In order to explore these hypotheses, we conducted a retrospective study, nested within a longitudinal prospective cohort study, evaluating the impact of low maternal vitamin D on congenital and peri/postnatal acquisition of CMV among HIV-infected, non-breastfeeding women and their HIV exposed but negative infants born between 1988 through 2015 at the Maternal, Child and Adolescent/Adult Center for Infectious Diseases and Virology (MCA) at the LAC+USC Medi- cal Center, in Los Angeles, California. PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 2 / 15 Low vitamin D linked to congenital CMV Fig 1. Study cohort based on infant CMV testing results. https://doi.org/10.1371/journal.pone.0228900.g001 Methods Study design and participants MCA is a comprehensive HIV clinic, serving women and their families. It is Los Angeles County’s largest referral site for HIV-infected pregnant women and their children, and cares for those who are under or uninsured. Informed consent was obtained for mothers and their newborns receiving care at MCA to participate in the University of Southern California Health Sciences Institutional Review Board–approved Natural History Study. The cohort design and participant selection for the current study are summarized in Fig 1. Mother-infant pairs were eligible for inclusion in this study if 1) they were both enrolled in MCA’s Natural History Study, 2) the mother was HIV-infected with evidence of CMV infection prior to the birth of the child, 3) the infant was HIV uninfected and had CMV testing between the ages of 0 to 6 months, and 4) stored maternal plasma obtained during pregnancy was available for vitamin D analysis. Among 559 mother-infant pairs with infant CMV testing between birth and age 6 months, 366 mothers had stored plasma available for vitamin D testing. Among these women, 340 had evidence of CMV infection: 312 were CMV seropositive and 28 with missing CMV PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 3 / 15 Low vitamin D linked to congenital CMV results, had infants who were CMV IgG+ at or near the time of birth. This was considered to be a transfer of maternal antibody and thus these mothers were considered CMV+ and included in the cohort. CMV testing in HIV-exposed infants was done as part of MCA’s routine care or as part of other MCA research studies. This testing included culture of urine and oral swabs, and poly- merase chain reaction (PCR) studies of blood. Cultures were performed using standard viro- logic methods of either shell-vial or tube cultures. PCR was performed by contracted send out laboratories (Quest Diagnostics, Focus Diagnostics) or in some cases, PCR was performed for other research studies by the MCA Laboratory. CMV tests available for analysis in this study included 1,101 urine cultures from 312 infants, 478 oral cultures from 158 infants, and 67 CMV blood PCR results from 55 infants. Congenital CMV was defined as 1 or more positive CMV tests between the ages of birth to 21 days. Peri/postnatal infection was defined as 1 or more positive results between 22 days to 6 months without a positive test during the congenital period. Vitamin D testing Stored maternal plasma samples were used for evaluation of vitamin D. Vitamin D is very sta- ble and samples may be frozen indefinitely and can withstand several freeze-thaw cycles with- out impacting results.[21] Specimens obtained at the time closest to the infant’s birth were used, with 50% obtained on the day of birth, 40% within 7 days of birth, 6.5% in the third tri- mester but before 7 days of birth, 3% in the second trimester, and 0.5% in the first trimester. Plasma samples were collected and stored in tubes containing either Ethylenediaminetetraace- tic acid (EDTA), heparin, or Acid Citrate Dextrose (ACD). Samples were sent to Pan Labora- tories, Irvine, CA for Vitamin D testing. Measurement of total 25(OH)D was performed by immunoassay (IDS ELISA, AC-57) using kits from Immuno-Diagnostic Systems (IDS), Phoe- nix, per package insert. The assay has a cross reactivity of 75% with 25 hydroxyvitamin D2. The assay has a sensitivity of 2.5 ng/ml and total interassay variation <8.7%.[22] Total calci- triol (1,25(OH)D ) was measured by immunoassay (IDS ELISA, AC-62), using kits from IDS. Calcitriol (1,25(OH)D ) was first purified by immuno-affinity purification and immunoassay was performed to quantitate calcitriol. The assay has a sensitivity of 2.5 pg/ml and total interas- say variation is <15%.[23] Sufficient 25(OH)D levels were defined as�32 ng/mL, 80 nmol/L, insufficient as 21–31 ng/mL, 52.5–77 nmol/L, inadequate as 11–20 ng/mL, 27.5–50 nmol/L, and deficient as�10 ng/mL, 25 nmol/L. Statistical analysis Demographic and HIV related variables evaluated included maternal age, race, ethnicity, HIV viral load categorized as <400 or�400 copies per ml, CD4 cell count categorized as <200 or �200 cells/mm , mode of delivery, infant gender, season, and antiretroviral treatment (ART). ART was further defined as 1) non-highly active antiretroviral therapy (non-HAART) which included women not taking any ART as well as those taking a single, dual, or a combination of agents felt to be less potent based on current standards; 2) HAART with a protease inhibitor; and 3) HAART without a protease inhibitor. For the purpose of this study, 25(OH)D levels were defined as sufficient if�32 ng/mL, 80 nmol/L. This cut point was selected based on prior research on optimal calcium homeostasis and bone health, recent clinical studies among pregnant women, and clinical applicability. [24,25] Additionally, data were divided into tertiles (rounded to the nearest 1 ng/ml) with 32 ng/mL defining the highest tertile. Sufficiency cut-points for 1,25(OH)D are less well estab- lished, therefore, tertiles (rounded to the nearest 1 pg/ml) based on the frequency distribution PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 4 / 15 Low vitamin D linked to congenital CMV were used in analyses. Both 1,25(OH)D and 25(OH)D were also evaluated as continuous vari- ables. Variables associated with vitamin D levels were identified using linear regression models with either 25(OH)D or 1,25(OH)D as the dependent variable and demographic and clinical characteristics as the independent variables. Generalized estimating equations (GEE) were used to account for the correlation among mothers with multiple birth outcomes. Correlates of 25(OH)D and 1,25(OH)D were analyzed separately. Univariate analyses of CMV infection were conducted using GEE logistic regression models with infant CMV status (positive/negative) as the outcome variable. Levels of 25(OH)D, 1,25 (OH)D and demographic and clinical characteristics were tested for their association with congenital CMV and peri/postnatal CMV in separate models GEE logistic regression models were used in multivariate modeling of CMV. Separate mod- els for 25(OH)D and 1,25(OH)D were analyzed for those with congenital CMV test results but only 1,25(OH)D for those with peri/postnatal results as no univariate relationship between 25(OH)D and peri/postnatal CMV infection was found. Factors associated with vita- min D and/or CMV in univariate models with P-value <0.15 were initially included in the multivariate models. Backward elimination was used to remove variables not associated with CMV (P>0.05). Final multivariate models controlled for CD4 count and HIV viral load due to their presumed impact on both vitamin D levels and CMV transmission. In the congenital group, covariates analyzed included ART category, race, ethnicity, HIV viral load, CD4 count, CD4 nadir during pregnancy, season, and maternal age at collection. In the peri/postnatal group, the covariates included ART category, race, ethnicity, HIV viral load, CD4 count, sea- son, and mode of delivery (vaginal versus caesarian birth). Ethics statement This study was approved by the University of Southern California’s Office for the Protection of Research Subjects, Health Sciences IRB. This was a retrospective study using data from an on- going, IRB approved Natural History Study, for which patients had signed consent. The cur- rent study posed no more than minimal risk and need for consent was waived. Results Among the entire cohort of 340 women/infant pairs, 38 infants had a positive CMV test between the age of birth and 6 months (11%). All positive infant test results are listed in S1 Table. Among the 300 infants with CMV testing between birth and 3 weeks, 14 or 4.7% were congenitally infected. Among the 315 infants with a CMV test between 22 days and 6 months (excluding the congenital cases), 24 infants or 7.6% were peri/postnatally infected. Among these 24 infants, 54% had a first positive test between age 4 and 10 weeks of life, 8% between weeks 11–17, and 38% between 18 and 26 weeks. Only 1 of these 24 infants was not tested in the congenital period while the other 23 had at least 1 negative test during the congenital period. Study cohort characteristics are summarized in Table 1 and are categorized based on infant CMV transmission category. The majority of women in the cohort self-identified as Hispanic white (73%) and were between the ages of 20 and 34 years. Most (80%) had HIV viral loads <400 copies/ml, 40% had CD4 counts above 500 cells/mm , and 78.5% were on HAART regimens. Maternal vitamin D levels are described in Table 1 and Fig 2A and 2B. Overall, two-thirds of the women had low vitamin D with 25(OH)D levels <32 ng/ml. Forty-two percent had insufficient 25(OH)D levels between 21–31 ng/ml, 24% had inadequate levels of 11–20 ng/ml, PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 5 / 15 Low vitamin D linked to congenital CMV Table 1. Study cohort characteristics. Variables Total sample (N = 340) Congenital CMV+ (N = 14) Peri/postnatal CMV+ (N = 24) CMV negative (N = 302) Maternal age (years) <20 30 (8.8%) 2 (14.3%) 2 (8.3%) 26 (8.6%) 20–34 240 (70.6%) 12 (85.7%) 17 (70.8%) 211 (69.9%) � 35 70 (20.6%) 0 5 (20.8%) 65 (21.5%) Race Hispanic white 248 (72.9%) 8 (57.1%) 22 (91.7%) 218 (72.2%) Non-Hispanic white 11 (3.2%) 1 (7.1%) 0 10 (3.3%) Black 77 (22.7%) 5 (35.7%) 0 72 (23.8%) Other 4 (1.2%) 0 2 (8.3%) 2 (0.7%) Plasma HIV-1 RNA (copies/mL) <400 266 (80.1%) 11 (84.6%) 21 (95.5%) 234 (78.8%) 400+ 66 (19.9%) 2 (15.4%) 1 (4.5%) 63 (21.2%) Data missing 8 1 2 5 CD4 cell count (cells/mm ) <200 39 (11.6%) 3 (21.4%) 3 (12.5%) 33 (11.0%) 200–500 163 (48.4%) 4 (28.6%) 10 (41.7%) 149 (49.8%) >500 135 (40.1%) 7 (50.0%) 11 (45.8%) 117 (39.1%) Data missing 3 0 0 3 ART regimen non-HAART 74 (22%) 2 (14.3%) 4 (16.7%) 68 (22.8%) HAART without PI 77 (22.9%) 5 (35.7%) 2 (8.3%) 70 (23.5%) HAART with PI 185 (55.6%) 7 (50%) 18 (75%) 160 (53.7%) Data missing 4 0 0 4 Mode of delivery Cesarean section 137 (40.9%) 4 (28.6%) 5 (21.7%) 128 (42.9%) Vaginal 198 (59.1%) 10 (71.4%) 18 (78.3%) 170 (57.1%) Data missing 5 0 1 4 Infant gender Female 155 (45.6%) 8 (57.1%) 13 (54.2%) 134 (44.4%) Male 185 (54.4%) 6 (42.9%) 11 (45.8%) 168 (55.6%) Season Summer/Fall 163 (47.9%) 5 (35.7%) 10 (41.7%) 148 (49%) Winter/Spring 177 (52.1%) 9 (64.3%) 14 (58.3%) 154 (51%) 25(OH)D levels, ng/ml <23 111 (32.7%) 3 (21.4%) 7 (29.2%) 101 (33.4%) 23–31 117 (34.4%) 9 (64.3%) 7 (29.2%) 101 (33.4%) � 32 112 (32.9%) 2 (14.3%) 10 (41.7%) 100 (33.1%) 1,25 (OH)D pg/ml median (IQR) 95.6 (69.8–125.4) 66.8 (54.9–73) 67.9 (57.9–80.5) 100 (73.5–128.9) � 74 116 (34.1%) 11 (78.6%) 17 (70.8%) 78 (25.8%) 75–115 118 (34.7%) 2 (14.3%) 4 (16.7%) 112 (37.1%) 116+ 106 (31.2%) 1 (7.1%) 3 (12.5%) 112 (37.1%) CD4, and HIV RNA levels tested at time point closest available to the vitamin D specimen test date non-HAART includes women on no ART, single agents, dual combinations, or any ART felt to be less active compared to current standards Abbreviations: ART, antiretroviral therapy; HAART, highly active antiretroviral therapy; IQR, Interquartile range; PI, protease-inhibitor; 25(OH)D, 25-hydroxyvitamin D; 1,25 (OH)D , 1,25-dihydroxyvitamin D https://doi.org/10.1371/journal.pone.0228900.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 6 / 15 Low vitamin D linked to congenital CMV Fig 2. a. The percentage of mothers with 25(OH)D levels above and below the cut point of 32 ng/ml compared by the CMV status of their infants. b. The percentage of mothers with low, middle or high 1,25-dihydroxyvitamin D levels, based on tertiles, compared by CMV transmission status. https://doi.org/10.1371/journal.pone.0228900.g002 and 2% were deficient with levels of�10 ng/ml (S2 Table). Maternal 25(OH)D and 1,25(OH) D levels were only weakly correlated (Spearman’s correlation coefficient = 0.21, P = 0.0001). Table 2 summarizes the variables associated with 25(OH)D and 1,25(OH)D . Maternal HIV viral load was significantly associated with both 25(OH)D (P = 0.002) and 1,25(OH)D (P = 0.02) with lower levels seen in women with higher viral loads. Lower 25(OH)D levels were associated with lower maternal CD4 cell counts (P = 0.05). Maternal ART category was significantly associated with 25(OH)D levels (P = 0.001) with the lowest values seen in the group on either no medications or non-HAART regimens. Season was significantly associated Table 2. Factors univariately associated with vitamin D levels. 25(OH)D ng/ml 1,25 (OH)D pg/ml � � Variables Mean (95%CI) P-value Mean (95% CI) P-value ART regimen 0.001 0.07 non-HAART 24.1 (21.6–26.6) 89.9 (80.5–99.4) HAART no PI 28.8 (26.6–30.9) 104.5 (95.0–113.9) HAART with PI 31.1 (29.3–32.9) 101.0 (95.5–106.6) Race 0.13 0.35 Hispanic white 28.9 (27.3–30.5) 101.3 (95.7–106.9) Non-Hispanic white 36.7 (28.4–44.9) 101.7 (77.8–125.6) Black 27.4 (24.7–30.1) 92.6 (84.5–100.8) Other 36.4 (28.3–44.6) 84.6 (41.7–127.6) Plasma HIV-1 RNA (copies/mL) 0.002 0.02 <400 30.1 (28.7–31.6) 102.5 (97.5–107.4) 400+ 24.8 (22.0–27.6) 89.1 (80.4–97.8) CD4 cell count (cells/mm ) 0.05 0.07 � 200 29.4 (27.9–30.9) 100.1 (95.3–104.9) <200 25.1 (21.4–28.8) 88.0 (76.5–99.5) Season 0.007 0.002 Summer & Fall 30.9 (28.7–33.0) 105.9 (99.3–112.6) Winter & Spring 27.2 (25.6–28.8) 92.8 (87.0–98.6) GEE linear regression model (separate models for 25(OH)D and 1,25 (OH)D ) CD4, and HIV RNA levels tested at time point closest available to the vitamin D specimen test date Least squares mean with 95% confidence interval Abbreviations: ART, antiretroviral therapy; HAART, highly active antiretroviral therapy; PI, protease inhibitor; 25 (OH)D, 25-hydroxyvitamin D; 1,25(OH)D , 1,25-dihydroxyvitamin D https://doi.org/10.1371/journal.pone.0228900.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 7 / 15 Low vitamin D linked to congenital CMV with both 25(OH)D (P = 0.007) and 1,25(OH)D (P = 0.002), with lower levels among women pregnant during months with lower levels of UV light. No relationships were found between CD8 cell counts nor CD4/CD8 ratios and either 25(OH)D or 1,25(OH)D . Factors associated with congenital transmission of CMV are summarized in Tables 3, 4 and 5. Lower levels of maternal bioactive vitamin D, 1,25(OH)D , were associated with increased congenital transmission of CMV in univariate (P = 0.006) and multivariate analyses (P = 0.009 for trend) controlling for maternal age, CD4 count, and HIV viral load categories. Women with 1,25(OH)D levels�74 pg/ml were 12 times more likely to transmit CMV congenitally to their infant (OR 12.2 [95% CI 1.61–92.2] P = 0.02) compared to women with levels in the high- est tertile of�116 pg/ml. In univariate analyses, women with 25(OH)D levels in the middle range of 23–31 ng/dl were more likely to transmit congenital CMV (OR 4.93 [1.05–23.1] P = 0.04) compared to women with levels�32. Borderline significance for this association was observed in the multi- variate analysis when controlling for maternal age, CD4 count, and HIV viral load categories (OR 4.88 [95% CI 0.99–24.1] P = 0.05). However, there was no significant difference found when comparing the lowest and highest tertile groups, nor when a cut point for vitamin D suf- ficiency of 32 ng/ml was used. Younger maternal age was also associated with increased risk of congenital CMV infection in univariate analysis (OR 0.90 [95% CI 0.83–0.98] P = 0.01) and remained significant in the multivariate model (OR 0.90 [95% CI 0.83–0.99] P = 0.03). Factors associated with peri/postnatal CMV infection status in both univariate and multi- variate analyses are summarized in Tables 3 and 6. In univariate analyses, only 1,25(OH)D was associated with peri/postnatal CMV infection (P = 0.0002). In addition, vaginal delivery compared to cesarean section was marginally associated with increased odds of peri/postnatal transmission. However, in multivariate analyses controlling for HIV viral load categories, CD4 count, 1,25(OH)D and ART, vaginal deliveries had 3 times greater odds of peri/postnatal transmission (OR 3.03 [95% CI 1.08–8.50] P = 0.04). Levels of 1,25(OH)D remained signifi- cant in multivariate analyses with higher maternal 1,25(OH)D levels protective for peri/post- partum CMV. Women with levels� 74 pg/ml had nearly 10 times greater odds peri/ postpartum transmission compared to women with 1,25(OH)D levels� 116 pg/ml control- ling for mode of delivery, CD4 and HIV viral load categories, and ART (OR 9.84 [95% CI 2.63–36.8] P = 0.003). There was no significant association seen between maternal 25(OH)D status and peri/postnatal CMV. Discussion This is the first study to evaluate the impact of maternal vitamin D status on congenital and peri/postnatal CMV transmission. In this study, lower levels of the bioactive form of vitamin D, calcitriol (1,25(OH)D ), were associated with increased odds of both congenital and peri/ postpartum CMV infections among perinatally exposed but HIV uninfected infants born to non-breastfeeding women with HIV. As seen in other cohorts, low levels of vitamin D were associated with lower maternal CD4 counts and higher HIV viral loads.[26–29] This supports the notion that vitamin D is involved in the immune functions related to HIV infection. How- ever, the relationships found between calcitriol and CMV transmission outcomes in this study were independent of these factors. There is growing evidence suggesting the importance of vitamin D in placental-related functions including placental development and implantation, calcium transport, immuno- modulatory functions, as well as an association between low calcitriol levels and pregnancy induced hypertension. [15,30–33] During pregnancy, non-classical, extra-renal production of 1,25(OH)D occurs in the placenta. In fact, healthy pregnancy is associated with a doubling or PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 8 / 15 Low vitamin D linked to congenital CMV Table 3. Factors univariately associated with congenital and perinatal/postnatal CMV transmission. Congenital CMV Perinatal/Postnatal CMV Variable OR (95% CI) P-value OR (95% CI) P-value 25(OH)D (ng/mL) 0.99 (0.94–1.04) 0.59 1.01 (0.99–1.04) 0.35 25(OH)D (ng/mL) 0.07 0.61 � 32 1.00 1.00 23–31 4.93 (1.05–23.1) 0.04 0.65 (0.24–1.78) 0.40 <23 1.69 (0.28–10.3) 0.57 0.66 (0.24–1.78) 0.41 1,25(OH)D (pg/mL) 0.97 (0.95–0.98) 0.0002 0.98 (0.97–0.99) 0.001 1,25(OH)D (pg/mL) 0.006 0.0002 � 116 1.00 1.00 75–115 2.01 (0.18–22.3) 0.57 1.32 (0.29–6.01) 0.72 � 74 12.6 (1.66–99.5) 0.01 7.88 (2.23–27.8) 0.001 ART Regimen 0.46 0.11 Non-HAART 1.00 1.00 HAART without PI 2.29 (0.43–12.2) 0.33 0.49 (0.09–2.72) 0.41 HAART with PI 1.18 (0.24–5.82) 0.84 2.03 (0.67–6.17) 0.21 Mode of delivery C-section 1.00 1.00 Vaginal 1.79 (0.55–5.80) 0.33 2.67 (0.97–7.34) 0.06 Baby gender Male 1.00 1.00 Female 1.66 (0.56–4.90) 0.36 1.55 (0.67–3.58) 0.30 Maternal Age 0.90 (0.83–0.98) 0.01 0.99 (0.94–1.05) 0.81 � � Race Non-Black 1.00 Black 2.03 (0.67–6.17) 0.21 Ethnicity Non-Hispanic 1.00 1.00 Hispanic 0.42 (0.14–1.23) 0.11 8.43 (1.12–63.5) 0.04 Plasma HIV-1 RNA (copies/mL) <400 1.00 1.00 400+ 0.80 (0.17–3.74) 0.78 0.18 (0.02–1.28) 0.09 CD4 Nadir During Pregnancy 200+ 1.00 1.00 <200 1.72 (0.52–5.71) 0.37 1.03 (0.37–2.88) 0.95 CD4 Cell Count (copies/mL) 200+ 1.00 1.00 <200 2.31 (0.61–8.74) 0.22 1.08 (0.30–3.90) 0.90 Season Summer/Fall 1.00 1.00 Winter/Spring 1.72 (0.57–5.22) 0.34 1.31 (0.56–3.04) 0.53 Modeled as a continuous variable � � Race was not included in the perinatal/postnatal model because no Black infants were perinatal/postnatal CMV+ GEE logistic regression model Abbreviations: ART, antiretroviral therapy; CI, confidence interval; HAART, highly active antiretroviral therapy; OR, odds ratio; PI, protease inhibitor; 1,25(OH)D2, 1,25-dihydroxyvitamin D; 25(OH)D, 25-hydroxyvitamin D https://doi.org/10.1371/journal.pone.0228900.t003 PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 9 / 15 Low vitamin D linked to congenital CMV Table 4. Factors associated with congenital CMV in multivariate model A. CMV Variables OR (95% CI) P-value P-value 1,25(OH)D (pg/mL) 0.01 0.009 � 116 1.00 75–115 2.18 (0.20–23.6) 0.52 � 74 12.2 (1.61–92.2) 0.02 Plasma HIV-1 RNA (copies/mL) <400 1.00 400+ 0.41 (0.08–2.00) 0.27 CD4 Cell Count (copies/mL) 200+ 1.00 <200 2.07 (0.42–10.3) 0.37 Maternal age 0.90 (0.83–0.99) 0.03 P-value for trend GEE logistic regression model Abbreviations: CI, confidence interval; OR, odds ratio; 1,25(OH)D2, 1,25-dihydroxyvitamin D https://doi.org/10.1371/journal.pone.0228900.t004 tripling of pre-pregnancy 1,25(OH)D levels while 25(OH)D levels typically remain unchanged.[25,34] This increase occurs without impacting serum or urinary calcium levels, demonstrating an uncoupling of vitamin D metabolism from the usual calcium-parathyroid hormone axis control.[33,34] Placental trophoblasts and maternal decidua actively convert vitamin D to its active form, 1,25(OH)D , in an intracrine manner.[32] Decidual and placental cells also contain an abundance of the vitamin D receptor (VDR), a ligand-activated transcrip- tion factor that controls the expression of over a thousand genes, allowing for a localized response when 1,25(OH)D binds. This response involves activation of the innate immune sys- tem including a dose-dependent production of the antimicrobial peptide, cathelicidin, ulti- mately protecting placental cells from infection and death.[18,35] It can be postulated that the Table 5. Factors associated with congenital CMV in multivariate model B. CMV Variables OR (95% CI) P-value P-value 25(OH)D (ng/mL) 0.10 0.46 � 32 1.00 23–31 4.88 (0.99–24.1) 0.05 <23 1.87 (0.27–12.9) 0.53 Plasma HIV-1 RNA (copies/mL) <400 1.00 400+ 0.42 (0.06–3.04) 0.39 CD4 Cell Count (copies/mL) 200+ 1.00 <200 3.08 (0.53–18.0) 0.21 Maternal age 0.90 (0.82–0.98) 0.02 P-value for trend GEE logistic regression model Abbreviations: CI, confidence interval; OR, odds ratio; 25(OH)D, 25-hydroxyvitamin D https://doi.org/10.1371/journal.pone.0228900.t005 PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 10 / 15 Low vitamin D linked to congenital CMV Table 6. Factors associated with peri/postnatal CMV in multivariate model. CMV Variables OR (95% CI) P-value P-value 1,25(OH)D (pg/mL) 0.0003 0.0008 � 116 1.00 75–115 1.60 (0.34–7.50) 0.55 � 74 9.84 (2.63–36.8) 0.0007 Plasma HIV-1 RNA (copies/mL) <400 1.00 400+ 2.18 (0.65–7.32) 0.21 CD4 Cell Count (copies/mL) 200+ 1.00 <200 1.25 (0.57–2.76) 0.58 ART Regimen 0.05 Non-HAART 1.00 HAART no PI 0.83 (0.10–7.25) 0.87 HAART with PI 4.21 (0.75–23.8) 0.10 Mode of delivery C-section 1.00 Vaginal 3.03 (1.08–8.50) 0.04 P-value for trend GEE logistic regression model Abbreviations: ART, antiretroviral therapy; CI, confidence interval; HAART, highly active antiretroviral therapy; OR, odds ratio; PI, protease inhibitor; 1,25(OH)D2, 1,25-dihydroxyvitamin D; 25(OH)D, 25-hydroxyvitamin D https://doi.org/10.1371/journal.pone.0228900.t006 additional 1,25(OH)D available to the developing fetus is in excess of the amount required for fetal skeletal development and instead may be essential for fetal and placental immunologic and antimicrobial functions. Our findings support this notion suggesting that the increased 1,25(OH)D available during pregnancy could aid immune functions important in blocking transplacental CMV transmission and possibly decrease maternal CMV shedding, thereby reducing congenital and peri/postnatal CMV infections. A prior study of untreated HIV infected pregnant women in Tanzania demonstrated increased mother-to-child-transmission of HIV in women with 25(OH)D levels <32 ng/ml. [24] In the current study, we found only weak evidence that lower levels of 25(OH)D, the main circulating form of vitamin D, may be associated with congenital CMV transmission. There was no trend as only women with levels in the middle tertile appeared to have increased CMV transmission compared to those with the highest levels, and there was no difference when comparing those with the lowest and highest levels, nor when the cut point for vitamin D sufficiency of 32 ng/ml was used for analysis. A randomized controlled trial of vitamin D supplementation in HIV negative pregnant women by Hollis, et al, demonstrated that a serum 25(OH)D level of 40 ng/ml was needed in order to achieve the supraphysiological increase in the bioactive form of vitamin D, calcitriol (1,25(OH)D ), during normal pregnancy.[25] It is possible a larger sample size is needed to similarly find a threshold serum value of 25(OH)D required to overcome any problems with placental dysfunction, supporting the necessary boost in calcitriol, and thereby aiding the important immunologic protective functions of the placenta. This study had several limitations. This is a retrospective study and the cohort was created, in part, based on availability of stored samples. This may have created inadvertent bias in PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 11 / 15 Low vitamin D linked to congenital CMV cohort selection. The women in this cohort were all considered seropositive for CMV based on maternal IgG, or early infant IgG in a few cases. Therefore, it can be assumed that the trans- missions during the congenital period were all non-primary infections due to reactivation of latent maternal CMV, or theoretically, reinfection with a different strain. Further study is needed to determine if vitamin D plays a role in low-seroprevalence populations as well, where higher rates of maternal primary CMV infection are more likely. The retrospective study design also limited the ability to do additional or confirmatory CMV PCR testing on infant urine, saliva or blood, due to the lack of availability of stored specimens. The results of this study were based mainly on culture results, as only 55 of the 340 infants had blood PCR test results, and there were no PCR results for urine or saliva. CMV cultures are less sensitive than PCR for detecting CMV and therefore the actual prevalence of congenital and peri/postnatal CMV infections may be underrepresented. More work is needed to demonstrate if the associa- tion between calcitriol, the active form of vitamin D, and congenital and early postnatal trans- mission of CMV found in this population of HIV-infected women is true in HIV uninfected women as well. Additionally, this study did not evaluate VDR expression nor the multitude of polymorphisms associated with altered VDR expression and function. It is unclear how the VDR and therefore the downstream effects of vitamin D may be impacted by CMV infection itself. In fact, a recent in vitro study demonstrated that the presence of CMV inhibited the expression of vitamin D receptors in fibroblasts.[36] Further study is needed to clarify the interplay between vitamin D and CMV infection and how this relationship may be important in protecting the developing fetus from potential infection. Although our study demonstrates an association between lower calcitriol (1,25(OH)D ) lev- els and increased CMV transmission congenitally and in the early postnatal period, we cannot comment on causation. It is unknown if the CMV and/or HIV infections deplete vitamin D as it is used in high demand in conjunction with fighting these infections, or if low maternal vita- min D leads to increased susceptibility to infection and/or increased or prolonged viral shed- ding. HIV and CMV infections are both known to cause significant placental damage and dysfunction and this could impact the placental production of calcitriol (1,25(OH)D ), poten- tially contributing to the lower levels seen in this study.[37–39] Additionally, the presence of pathogens may affect local calcitriol production. In vitro, the presence of HIV and LPS impacts the production and breakdown of calcitriol by affecting CYP27B1 and CYP24A1 gene expres- sion in monocytes.[40] Future, prospective studies are needed in HIV positive and negative populations to further clarify these complex relationships. If found to be causal and protective in prospective studies, targeted vitamin D supplementation, with the goal of supporting the necessary rise in calcitriol during pregnancy, could represent a safe and inexpensive tool in preventing CMV transmis- sion from mother to infant. Supporting information S1 Table. Number of positive CMV tests by test type for all CMV+ infants. (DOCX) S2 Table. 25-Hydroxyvitamin D sufficiency by infant transmission category. (DOCX) Acknowledgments The authors would like to acknowledge the MCA Laboratory staff for their assistance with this study. PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 12 / 15 Low vitamin D linked to congenital CMV Author Contributions Conceptualization: Allison Bearden, Kristi Van Winden. Data curation: Allison Bearden, Toni Frederick, Naoko Kono, Lorayne Barton. Formal analysis: Toni Frederick, Naoko Kono. Funding acquisition: Allison Bearden, Kristi Van Winden. Investigation: Allison Bearden, Kristi Van Winden, Raj Pandian. Methodology: Allison Bearden, Kristi Van Winden, Toni Frederick, Naoko Kono, Eva Oper- skalski, Andrea Kovacs. Project administration: Allison Bearden. Resources: Andrea Kovacs. Supervision: Allison Bearden, Alice Stek, Andrea Kovacs. Writing – original draft: Allison Bearden. 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Low maternal vitamin D is associated with increased risk of congenital and peri/postnatal transmission of Cytomegalovirus in women with HIV

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Copyright: © 2020 Bearden et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: We are unable to publicly share the dataset from this study due to ethical and legal restrictions. According the University of Southern California’s Office of Ethics and Compliance, there is no way to make the dataset truly de-identified. The dataset is made up solely of women with a diagnosis of HIV and their offspring. The diagnosis of HIV is an indirect identifier and considered sensitive information requiring additional protections. Additional indirect identifiers in the dataset include age, race/ethnicity, and sex. Additionally, the consent signed by the study participants did not include a provision for their data to be made publicly accessible. Data requests can be directed to department administrator, Carlota Obnillas, obnillas@usc.edu, and if the request is approved, the USC Stevens Center for Innovation will assist and create a Data Transfer Agreement. Funding: This work was supported by the SC CTSI (https://sc-ctsi.org), (NIH/NCRR/NCATS) through Grant UL1TR000130, awarded to AK and AB. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health. This study was funded in part by a seed grant provided by the Department of Obstetrics and Gynecology at the University of Southern California awarded to KV. RP received support in the form of a salary from Pan Laboratories, Irvine, CA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Competing interests: RP is the salaried Director of Pan Laboratories, Irvine, CA. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
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Abstract

transmission of Cytomegalovirus in women with HIV. PLoS ONE 15(2): e0228900. https://doi.org/ 10.1371/journal.pone.0228900 Background Editor: Michael Nevels, University of St Andrews, CMV infection of the fetus or neonate can lead to devastating disease, and there are no UNITED KINGDOM effective prevention strategies to date. Vitamin D is a potent immunomodulator, supports Received: October 15, 2019 antiviral immune responses, and plays an important role in placental immunity. Accepted: January 24, 2020 Published: February 13, 2020 Methods Peer Review History: PLOS recognizes the Retrospective cohort study to evaluate the impact of low maternal vitamin D on congenital benefits of transparency in the peer review and early postnatal transmission of CMV among HIV-infected, non-breastfeeding women process; therefore, we enable the publication of and their HIV exposed but negative infants from an urban HIV clinic. Vitamin D panel was all of the content of peer review and author responses alongside final, published articles. The performed on stored maternal plasma obtained near time of delivery. Infant CMV testing at editorial history of this article is available here: 0–6 months included urine and oral cultures, and/or serum polymerase chain reaction https://doi.org/10.1371/journal.pone.0228900 testing. Copyright:© 2020 Bearden et al. This is an open access article distributed under the terms of the Results Creative Commons Attribution License, which permits unrestricted use, distribution, and Cohort included 340 mother-infant pairs (births 1991–2014). Among 38 infants (11%) with a reproduction in any medium, provided the original CMV+ test between 0–6 months, 4.7% (14/300) had congenital CMV transmission (CMV+ author and source are credited. test 0–3 weeks), and 7.6% (24/315) had peri/postnatal CMV (CMV+ test>3 weeks-6 Data Availability Statement: We are unable to months). Women with lower calcitriol (1,25-dihydroxyvitamin D), the active form of vitamin publicly share the dataset from this study due to ethical and legal restrictions. According the D, were more likely to have an infant with congenital (OR 12.2 [95% CI 1.61–92.2] P = 0.02) PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 1 / 15 Low vitamin D linked to congenital CMV University of Southern California’s Office of Ethics and peri/postnatal (OR 9.84 [95% CI 2.63–36.8] P = 0.0007) infections in multivariate analy- and Compliance, there is no way to make the ses, independent of maternal HIV viral load and CD4 count. dataset truly de-identified. The dataset is made up solely of women with a diagnosis of HIV and their offspring. The diagnosis of HIV is an indirect Conclusion identifier and considered sensitive information requiring additional protections. Additional indirect This study demonstrates an association between inadequate maternal calcitriol during preg- identifiers in the dataset include age, race/ethnicity, nancy and increased congenital and early postnatal acquisition of CMV among non-breast- and sex. Additionally, the consent signed by the feeding women with HIV and their HIV negative infants. study participants did not include a provision for their data to be made publicly accessible. Data requests can be directed to department administrator, Carlota Obnillas, obnillas@usc.edu, and if the request is approved, the USC Stevens Center for Innovation will assist and create a Data Transfer Agreement. Introduction Funding: This work was supported by the SC CTSI Human Cytomegalovirus (CMV) infection is the most common congenital infection world- (https://sc-ctsi.org), (NIH/NCRR/NCATS) through wide. It is the top non-genetic cause of childhood deafness in the world and can lead to neuro- Grant UL1TR000130, awarded to AK and AB. Its logic and neurodevelopmental disorders, multisystem illness, growth and development contents are solely the responsibility of the authors abnormalities, and death.[1,2] Approximately 50–70% of women of childbearing age in devel- and do not necessarily represent the official views oped countries are CMV infected, with the highest prevalence among women of lower socio- of the National Institutes of Health. This study was funded in part by a seed grant provided by the economic status.[3] Seroprevalence approaches 100% among women of child-bearing age in Department of Obstetrics and Gynecology at the resource-limited countries and in those with Human Immunodeficiency Virus (HIV) infec- University of Southern California awarded to KV. tion.[2,4] Mother-to-child-transmission (MTCT) of CMV can occur prenatally (congenital RP received support in the form of a salary from infection), during birth, and postnatally through breast milk.[5] Mothers and other caregivers Pan Laboratories, Irvine, CA. The funders had no can also transmit CMV to their infants postnatally through infected secretions.[2] Maternal role in study design, data collection and analysis, decision to publish, or preparation of the CMV infections and reactivations are often asymptomatic and unnoticed, and unlike HIV, manuscript. The specific roles of these authors are there are currently no effective strategies widely implemented for the prevention of MTCT of articulated in the ‘author contributions’ section. CMV.[6] Rates of congenital CMV are often higher among infants of women with HIV infec- Competing interests: RP is the salaried Director of tion, making them an ideal population for study.[2,4,7–11] Pan Laboratories, Irvine, CA. There are no patents, Vitamin D is obtained either from exposure to ultraviolet light or from the diet. In addition products in development or marketed products to to its role in calcium homeostasis and skeletal health, Vitamin D is a well-known and potent declare. This does not alter our adherence to PLOS modulator of the immune system.[12] Vitamin D supports immune system antiviral responses ONE policies on sharing data and materials. through the induction of autophagy and production of antimicrobial peptides like cathelicidin, and likely plays an important role in helping to protect the developing fetus from infections during pregnancy.[13–16] A multitude of cells in the body have the vitamin D receptor and many cells, including the cells of the placenta, also have the ability to convert 25-hydroxyvita- min D (25(OH)D), the main circulating form of vitamin D, to its bioactive form, 1,25-dihy- droxyvitamin D (1,25(OH)D ).[17,18] This allows for local production of 1,25(OH)D and the 2 2 subsequent vitamin D-dependent antimicrobial immune responses in the setting of specific conditions or stimuli.[15,18–20] Vitamin D’s important role in supporting the immune system’s antiviral functions, includ- ing those at the level of the placenta, suggests its relevance to MTCT of CMV in utero. Addi- tionally, vitamin D may contribute to the immune system’s ability to limit viral shedding and therefore play a role in limiting perinatal and early postnatal CMV transmission. In order to explore these hypotheses, we conducted a retrospective study, nested within a longitudinal prospective cohort study, evaluating the impact of low maternal vitamin D on congenital and peri/postnatal acquisition of CMV among HIV-infected, non-breastfeeding women and their HIV exposed but negative infants born between 1988 through 2015 at the Maternal, Child and Adolescent/Adult Center for Infectious Diseases and Virology (MCA) at the LAC+USC Medi- cal Center, in Los Angeles, California. PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 2 / 15 Low vitamin D linked to congenital CMV Fig 1. Study cohort based on infant CMV testing results. https://doi.org/10.1371/journal.pone.0228900.g001 Methods Study design and participants MCA is a comprehensive HIV clinic, serving women and their families. It is Los Angeles County’s largest referral site for HIV-infected pregnant women and their children, and cares for those who are under or uninsured. Informed consent was obtained for mothers and their newborns receiving care at MCA to participate in the University of Southern California Health Sciences Institutional Review Board–approved Natural History Study. The cohort design and participant selection for the current study are summarized in Fig 1. Mother-infant pairs were eligible for inclusion in this study if 1) they were both enrolled in MCA’s Natural History Study, 2) the mother was HIV-infected with evidence of CMV infection prior to the birth of the child, 3) the infant was HIV uninfected and had CMV testing between the ages of 0 to 6 months, and 4) stored maternal plasma obtained during pregnancy was available for vitamin D analysis. Among 559 mother-infant pairs with infant CMV testing between birth and age 6 months, 366 mothers had stored plasma available for vitamin D testing. Among these women, 340 had evidence of CMV infection: 312 were CMV seropositive and 28 with missing CMV PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 3 / 15 Low vitamin D linked to congenital CMV results, had infants who were CMV IgG+ at or near the time of birth. This was considered to be a transfer of maternal antibody and thus these mothers were considered CMV+ and included in the cohort. CMV testing in HIV-exposed infants was done as part of MCA’s routine care or as part of other MCA research studies. This testing included culture of urine and oral swabs, and poly- merase chain reaction (PCR) studies of blood. Cultures were performed using standard viro- logic methods of either shell-vial or tube cultures. PCR was performed by contracted send out laboratories (Quest Diagnostics, Focus Diagnostics) or in some cases, PCR was performed for other research studies by the MCA Laboratory. CMV tests available for analysis in this study included 1,101 urine cultures from 312 infants, 478 oral cultures from 158 infants, and 67 CMV blood PCR results from 55 infants. Congenital CMV was defined as 1 or more positive CMV tests between the ages of birth to 21 days. Peri/postnatal infection was defined as 1 or more positive results between 22 days to 6 months without a positive test during the congenital period. Vitamin D testing Stored maternal plasma samples were used for evaluation of vitamin D. Vitamin D is very sta- ble and samples may be frozen indefinitely and can withstand several freeze-thaw cycles with- out impacting results.[21] Specimens obtained at the time closest to the infant’s birth were used, with 50% obtained on the day of birth, 40% within 7 days of birth, 6.5% in the third tri- mester but before 7 days of birth, 3% in the second trimester, and 0.5% in the first trimester. Plasma samples were collected and stored in tubes containing either Ethylenediaminetetraace- tic acid (EDTA), heparin, or Acid Citrate Dextrose (ACD). Samples were sent to Pan Labora- tories, Irvine, CA for Vitamin D testing. Measurement of total 25(OH)D was performed by immunoassay (IDS ELISA, AC-57) using kits from Immuno-Diagnostic Systems (IDS), Phoe- nix, per package insert. The assay has a cross reactivity of 75% with 25 hydroxyvitamin D2. The assay has a sensitivity of 2.5 ng/ml and total interassay variation <8.7%.[22] Total calci- triol (1,25(OH)D ) was measured by immunoassay (IDS ELISA, AC-62), using kits from IDS. Calcitriol (1,25(OH)D ) was first purified by immuno-affinity purification and immunoassay was performed to quantitate calcitriol. The assay has a sensitivity of 2.5 pg/ml and total interas- say variation is <15%.[23] Sufficient 25(OH)D levels were defined as�32 ng/mL, 80 nmol/L, insufficient as 21–31 ng/mL, 52.5–77 nmol/L, inadequate as 11–20 ng/mL, 27.5–50 nmol/L, and deficient as�10 ng/mL, 25 nmol/L. Statistical analysis Demographic and HIV related variables evaluated included maternal age, race, ethnicity, HIV viral load categorized as <400 or�400 copies per ml, CD4 cell count categorized as <200 or �200 cells/mm , mode of delivery, infant gender, season, and antiretroviral treatment (ART). ART was further defined as 1) non-highly active antiretroviral therapy (non-HAART) which included women not taking any ART as well as those taking a single, dual, or a combination of agents felt to be less potent based on current standards; 2) HAART with a protease inhibitor; and 3) HAART without a protease inhibitor. For the purpose of this study, 25(OH)D levels were defined as sufficient if�32 ng/mL, 80 nmol/L. This cut point was selected based on prior research on optimal calcium homeostasis and bone health, recent clinical studies among pregnant women, and clinical applicability. [24,25] Additionally, data were divided into tertiles (rounded to the nearest 1 ng/ml) with 32 ng/mL defining the highest tertile. Sufficiency cut-points for 1,25(OH)D are less well estab- lished, therefore, tertiles (rounded to the nearest 1 pg/ml) based on the frequency distribution PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 4 / 15 Low vitamin D linked to congenital CMV were used in analyses. Both 1,25(OH)D and 25(OH)D were also evaluated as continuous vari- ables. Variables associated with vitamin D levels were identified using linear regression models with either 25(OH)D or 1,25(OH)D as the dependent variable and demographic and clinical characteristics as the independent variables. Generalized estimating equations (GEE) were used to account for the correlation among mothers with multiple birth outcomes. Correlates of 25(OH)D and 1,25(OH)D were analyzed separately. Univariate analyses of CMV infection were conducted using GEE logistic regression models with infant CMV status (positive/negative) as the outcome variable. Levels of 25(OH)D, 1,25 (OH)D and demographic and clinical characteristics were tested for their association with congenital CMV and peri/postnatal CMV in separate models GEE logistic regression models were used in multivariate modeling of CMV. Separate mod- els for 25(OH)D and 1,25(OH)D were analyzed for those with congenital CMV test results but only 1,25(OH)D for those with peri/postnatal results as no univariate relationship between 25(OH)D and peri/postnatal CMV infection was found. Factors associated with vita- min D and/or CMV in univariate models with P-value <0.15 were initially included in the multivariate models. Backward elimination was used to remove variables not associated with CMV (P>0.05). Final multivariate models controlled for CD4 count and HIV viral load due to their presumed impact on both vitamin D levels and CMV transmission. In the congenital group, covariates analyzed included ART category, race, ethnicity, HIV viral load, CD4 count, CD4 nadir during pregnancy, season, and maternal age at collection. In the peri/postnatal group, the covariates included ART category, race, ethnicity, HIV viral load, CD4 count, sea- son, and mode of delivery (vaginal versus caesarian birth). Ethics statement This study was approved by the University of Southern California’s Office for the Protection of Research Subjects, Health Sciences IRB. This was a retrospective study using data from an on- going, IRB approved Natural History Study, for which patients had signed consent. The cur- rent study posed no more than minimal risk and need for consent was waived. Results Among the entire cohort of 340 women/infant pairs, 38 infants had a positive CMV test between the age of birth and 6 months (11%). All positive infant test results are listed in S1 Table. Among the 300 infants with CMV testing between birth and 3 weeks, 14 or 4.7% were congenitally infected. Among the 315 infants with a CMV test between 22 days and 6 months (excluding the congenital cases), 24 infants or 7.6% were peri/postnatally infected. Among these 24 infants, 54% had a first positive test between age 4 and 10 weeks of life, 8% between weeks 11–17, and 38% between 18 and 26 weeks. Only 1 of these 24 infants was not tested in the congenital period while the other 23 had at least 1 negative test during the congenital period. Study cohort characteristics are summarized in Table 1 and are categorized based on infant CMV transmission category. The majority of women in the cohort self-identified as Hispanic white (73%) and were between the ages of 20 and 34 years. Most (80%) had HIV viral loads <400 copies/ml, 40% had CD4 counts above 500 cells/mm , and 78.5% were on HAART regimens. Maternal vitamin D levels are described in Table 1 and Fig 2A and 2B. Overall, two-thirds of the women had low vitamin D with 25(OH)D levels <32 ng/ml. Forty-two percent had insufficient 25(OH)D levels between 21–31 ng/ml, 24% had inadequate levels of 11–20 ng/ml, PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 5 / 15 Low vitamin D linked to congenital CMV Table 1. Study cohort characteristics. Variables Total sample (N = 340) Congenital CMV+ (N = 14) Peri/postnatal CMV+ (N = 24) CMV negative (N = 302) Maternal age (years) <20 30 (8.8%) 2 (14.3%) 2 (8.3%) 26 (8.6%) 20–34 240 (70.6%) 12 (85.7%) 17 (70.8%) 211 (69.9%) � 35 70 (20.6%) 0 5 (20.8%) 65 (21.5%) Race Hispanic white 248 (72.9%) 8 (57.1%) 22 (91.7%) 218 (72.2%) Non-Hispanic white 11 (3.2%) 1 (7.1%) 0 10 (3.3%) Black 77 (22.7%) 5 (35.7%) 0 72 (23.8%) Other 4 (1.2%) 0 2 (8.3%) 2 (0.7%) Plasma HIV-1 RNA (copies/mL) <400 266 (80.1%) 11 (84.6%) 21 (95.5%) 234 (78.8%) 400+ 66 (19.9%) 2 (15.4%) 1 (4.5%) 63 (21.2%) Data missing 8 1 2 5 CD4 cell count (cells/mm ) <200 39 (11.6%) 3 (21.4%) 3 (12.5%) 33 (11.0%) 200–500 163 (48.4%) 4 (28.6%) 10 (41.7%) 149 (49.8%) >500 135 (40.1%) 7 (50.0%) 11 (45.8%) 117 (39.1%) Data missing 3 0 0 3 ART regimen non-HAART 74 (22%) 2 (14.3%) 4 (16.7%) 68 (22.8%) HAART without PI 77 (22.9%) 5 (35.7%) 2 (8.3%) 70 (23.5%) HAART with PI 185 (55.6%) 7 (50%) 18 (75%) 160 (53.7%) Data missing 4 0 0 4 Mode of delivery Cesarean section 137 (40.9%) 4 (28.6%) 5 (21.7%) 128 (42.9%) Vaginal 198 (59.1%) 10 (71.4%) 18 (78.3%) 170 (57.1%) Data missing 5 0 1 4 Infant gender Female 155 (45.6%) 8 (57.1%) 13 (54.2%) 134 (44.4%) Male 185 (54.4%) 6 (42.9%) 11 (45.8%) 168 (55.6%) Season Summer/Fall 163 (47.9%) 5 (35.7%) 10 (41.7%) 148 (49%) Winter/Spring 177 (52.1%) 9 (64.3%) 14 (58.3%) 154 (51%) 25(OH)D levels, ng/ml <23 111 (32.7%) 3 (21.4%) 7 (29.2%) 101 (33.4%) 23–31 117 (34.4%) 9 (64.3%) 7 (29.2%) 101 (33.4%) � 32 112 (32.9%) 2 (14.3%) 10 (41.7%) 100 (33.1%) 1,25 (OH)D pg/ml median (IQR) 95.6 (69.8–125.4) 66.8 (54.9–73) 67.9 (57.9–80.5) 100 (73.5–128.9) � 74 116 (34.1%) 11 (78.6%) 17 (70.8%) 78 (25.8%) 75–115 118 (34.7%) 2 (14.3%) 4 (16.7%) 112 (37.1%) 116+ 106 (31.2%) 1 (7.1%) 3 (12.5%) 112 (37.1%) CD4, and HIV RNA levels tested at time point closest available to the vitamin D specimen test date non-HAART includes women on no ART, single agents, dual combinations, or any ART felt to be less active compared to current standards Abbreviations: ART, antiretroviral therapy; HAART, highly active antiretroviral therapy; IQR, Interquartile range; PI, protease-inhibitor; 25(OH)D, 25-hydroxyvitamin D; 1,25 (OH)D , 1,25-dihydroxyvitamin D https://doi.org/10.1371/journal.pone.0228900.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 6 / 15 Low vitamin D linked to congenital CMV Fig 2. a. The percentage of mothers with 25(OH)D levels above and below the cut point of 32 ng/ml compared by the CMV status of their infants. b. The percentage of mothers with low, middle or high 1,25-dihydroxyvitamin D levels, based on tertiles, compared by CMV transmission status. https://doi.org/10.1371/journal.pone.0228900.g002 and 2% were deficient with levels of�10 ng/ml (S2 Table). Maternal 25(OH)D and 1,25(OH) D levels were only weakly correlated (Spearman’s correlation coefficient = 0.21, P = 0.0001). Table 2 summarizes the variables associated with 25(OH)D and 1,25(OH)D . Maternal HIV viral load was significantly associated with both 25(OH)D (P = 0.002) and 1,25(OH)D (P = 0.02) with lower levels seen in women with higher viral loads. Lower 25(OH)D levels were associated with lower maternal CD4 cell counts (P = 0.05). Maternal ART category was significantly associated with 25(OH)D levels (P = 0.001) with the lowest values seen in the group on either no medications or non-HAART regimens. Season was significantly associated Table 2. Factors univariately associated with vitamin D levels. 25(OH)D ng/ml 1,25 (OH)D pg/ml � � Variables Mean (95%CI) P-value Mean (95% CI) P-value ART regimen 0.001 0.07 non-HAART 24.1 (21.6–26.6) 89.9 (80.5–99.4) HAART no PI 28.8 (26.6–30.9) 104.5 (95.0–113.9) HAART with PI 31.1 (29.3–32.9) 101.0 (95.5–106.6) Race 0.13 0.35 Hispanic white 28.9 (27.3–30.5) 101.3 (95.7–106.9) Non-Hispanic white 36.7 (28.4–44.9) 101.7 (77.8–125.6) Black 27.4 (24.7–30.1) 92.6 (84.5–100.8) Other 36.4 (28.3–44.6) 84.6 (41.7–127.6) Plasma HIV-1 RNA (copies/mL) 0.002 0.02 <400 30.1 (28.7–31.6) 102.5 (97.5–107.4) 400+ 24.8 (22.0–27.6) 89.1 (80.4–97.8) CD4 cell count (cells/mm ) 0.05 0.07 � 200 29.4 (27.9–30.9) 100.1 (95.3–104.9) <200 25.1 (21.4–28.8) 88.0 (76.5–99.5) Season 0.007 0.002 Summer & Fall 30.9 (28.7–33.0) 105.9 (99.3–112.6) Winter & Spring 27.2 (25.6–28.8) 92.8 (87.0–98.6) GEE linear regression model (separate models for 25(OH)D and 1,25 (OH)D ) CD4, and HIV RNA levels tested at time point closest available to the vitamin D specimen test date Least squares mean with 95% confidence interval Abbreviations: ART, antiretroviral therapy; HAART, highly active antiretroviral therapy; PI, protease inhibitor; 25 (OH)D, 25-hydroxyvitamin D; 1,25(OH)D , 1,25-dihydroxyvitamin D https://doi.org/10.1371/journal.pone.0228900.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 7 / 15 Low vitamin D linked to congenital CMV with both 25(OH)D (P = 0.007) and 1,25(OH)D (P = 0.002), with lower levels among women pregnant during months with lower levels of UV light. No relationships were found between CD8 cell counts nor CD4/CD8 ratios and either 25(OH)D or 1,25(OH)D . Factors associated with congenital transmission of CMV are summarized in Tables 3, 4 and 5. Lower levels of maternal bioactive vitamin D, 1,25(OH)D , were associated with increased congenital transmission of CMV in univariate (P = 0.006) and multivariate analyses (P = 0.009 for trend) controlling for maternal age, CD4 count, and HIV viral load categories. Women with 1,25(OH)D levels�74 pg/ml were 12 times more likely to transmit CMV congenitally to their infant (OR 12.2 [95% CI 1.61–92.2] P = 0.02) compared to women with levels in the high- est tertile of�116 pg/ml. In univariate analyses, women with 25(OH)D levels in the middle range of 23–31 ng/dl were more likely to transmit congenital CMV (OR 4.93 [1.05–23.1] P = 0.04) compared to women with levels�32. Borderline significance for this association was observed in the multi- variate analysis when controlling for maternal age, CD4 count, and HIV viral load categories (OR 4.88 [95% CI 0.99–24.1] P = 0.05). However, there was no significant difference found when comparing the lowest and highest tertile groups, nor when a cut point for vitamin D suf- ficiency of 32 ng/ml was used. Younger maternal age was also associated with increased risk of congenital CMV infection in univariate analysis (OR 0.90 [95% CI 0.83–0.98] P = 0.01) and remained significant in the multivariate model (OR 0.90 [95% CI 0.83–0.99] P = 0.03). Factors associated with peri/postnatal CMV infection status in both univariate and multi- variate analyses are summarized in Tables 3 and 6. In univariate analyses, only 1,25(OH)D was associated with peri/postnatal CMV infection (P = 0.0002). In addition, vaginal delivery compared to cesarean section was marginally associated with increased odds of peri/postnatal transmission. However, in multivariate analyses controlling for HIV viral load categories, CD4 count, 1,25(OH)D and ART, vaginal deliveries had 3 times greater odds of peri/postnatal transmission (OR 3.03 [95% CI 1.08–8.50] P = 0.04). Levels of 1,25(OH)D remained signifi- cant in multivariate analyses with higher maternal 1,25(OH)D levels protective for peri/post- partum CMV. Women with levels� 74 pg/ml had nearly 10 times greater odds peri/ postpartum transmission compared to women with 1,25(OH)D levels� 116 pg/ml control- ling for mode of delivery, CD4 and HIV viral load categories, and ART (OR 9.84 [95% CI 2.63–36.8] P = 0.003). There was no significant association seen between maternal 25(OH)D status and peri/postnatal CMV. Discussion This is the first study to evaluate the impact of maternal vitamin D status on congenital and peri/postnatal CMV transmission. In this study, lower levels of the bioactive form of vitamin D, calcitriol (1,25(OH)D ), were associated with increased odds of both congenital and peri/ postpartum CMV infections among perinatally exposed but HIV uninfected infants born to non-breastfeeding women with HIV. As seen in other cohorts, low levels of vitamin D were associated with lower maternal CD4 counts and higher HIV viral loads.[26–29] This supports the notion that vitamin D is involved in the immune functions related to HIV infection. How- ever, the relationships found between calcitriol and CMV transmission outcomes in this study were independent of these factors. There is growing evidence suggesting the importance of vitamin D in placental-related functions including placental development and implantation, calcium transport, immuno- modulatory functions, as well as an association between low calcitriol levels and pregnancy induced hypertension. [15,30–33] During pregnancy, non-classical, extra-renal production of 1,25(OH)D occurs in the placenta. In fact, healthy pregnancy is associated with a doubling or PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 8 / 15 Low vitamin D linked to congenital CMV Table 3. Factors univariately associated with congenital and perinatal/postnatal CMV transmission. Congenital CMV Perinatal/Postnatal CMV Variable OR (95% CI) P-value OR (95% CI) P-value 25(OH)D (ng/mL) 0.99 (0.94–1.04) 0.59 1.01 (0.99–1.04) 0.35 25(OH)D (ng/mL) 0.07 0.61 � 32 1.00 1.00 23–31 4.93 (1.05–23.1) 0.04 0.65 (0.24–1.78) 0.40 <23 1.69 (0.28–10.3) 0.57 0.66 (0.24–1.78) 0.41 1,25(OH)D (pg/mL) 0.97 (0.95–0.98) 0.0002 0.98 (0.97–0.99) 0.001 1,25(OH)D (pg/mL) 0.006 0.0002 � 116 1.00 1.00 75–115 2.01 (0.18–22.3) 0.57 1.32 (0.29–6.01) 0.72 � 74 12.6 (1.66–99.5) 0.01 7.88 (2.23–27.8) 0.001 ART Regimen 0.46 0.11 Non-HAART 1.00 1.00 HAART without PI 2.29 (0.43–12.2) 0.33 0.49 (0.09–2.72) 0.41 HAART with PI 1.18 (0.24–5.82) 0.84 2.03 (0.67–6.17) 0.21 Mode of delivery C-section 1.00 1.00 Vaginal 1.79 (0.55–5.80) 0.33 2.67 (0.97–7.34) 0.06 Baby gender Male 1.00 1.00 Female 1.66 (0.56–4.90) 0.36 1.55 (0.67–3.58) 0.30 Maternal Age 0.90 (0.83–0.98) 0.01 0.99 (0.94–1.05) 0.81 � � Race Non-Black 1.00 Black 2.03 (0.67–6.17) 0.21 Ethnicity Non-Hispanic 1.00 1.00 Hispanic 0.42 (0.14–1.23) 0.11 8.43 (1.12–63.5) 0.04 Plasma HIV-1 RNA (copies/mL) <400 1.00 1.00 400+ 0.80 (0.17–3.74) 0.78 0.18 (0.02–1.28) 0.09 CD4 Nadir During Pregnancy 200+ 1.00 1.00 <200 1.72 (0.52–5.71) 0.37 1.03 (0.37–2.88) 0.95 CD4 Cell Count (copies/mL) 200+ 1.00 1.00 <200 2.31 (0.61–8.74) 0.22 1.08 (0.30–3.90) 0.90 Season Summer/Fall 1.00 1.00 Winter/Spring 1.72 (0.57–5.22) 0.34 1.31 (0.56–3.04) 0.53 Modeled as a continuous variable � � Race was not included in the perinatal/postnatal model because no Black infants were perinatal/postnatal CMV+ GEE logistic regression model Abbreviations: ART, antiretroviral therapy; CI, confidence interval; HAART, highly active antiretroviral therapy; OR, odds ratio; PI, protease inhibitor; 1,25(OH)D2, 1,25-dihydroxyvitamin D; 25(OH)D, 25-hydroxyvitamin D https://doi.org/10.1371/journal.pone.0228900.t003 PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 9 / 15 Low vitamin D linked to congenital CMV Table 4. Factors associated with congenital CMV in multivariate model A. CMV Variables OR (95% CI) P-value P-value 1,25(OH)D (pg/mL) 0.01 0.009 � 116 1.00 75–115 2.18 (0.20–23.6) 0.52 � 74 12.2 (1.61–92.2) 0.02 Plasma HIV-1 RNA (copies/mL) <400 1.00 400+ 0.41 (0.08–2.00) 0.27 CD4 Cell Count (copies/mL) 200+ 1.00 <200 2.07 (0.42–10.3) 0.37 Maternal age 0.90 (0.83–0.99) 0.03 P-value for trend GEE logistic regression model Abbreviations: CI, confidence interval; OR, odds ratio; 1,25(OH)D2, 1,25-dihydroxyvitamin D https://doi.org/10.1371/journal.pone.0228900.t004 tripling of pre-pregnancy 1,25(OH)D levels while 25(OH)D levels typically remain unchanged.[25,34] This increase occurs without impacting serum or urinary calcium levels, demonstrating an uncoupling of vitamin D metabolism from the usual calcium-parathyroid hormone axis control.[33,34] Placental trophoblasts and maternal decidua actively convert vitamin D to its active form, 1,25(OH)D , in an intracrine manner.[32] Decidual and placental cells also contain an abundance of the vitamin D receptor (VDR), a ligand-activated transcrip- tion factor that controls the expression of over a thousand genes, allowing for a localized response when 1,25(OH)D binds. This response involves activation of the innate immune sys- tem including a dose-dependent production of the antimicrobial peptide, cathelicidin, ulti- mately protecting placental cells from infection and death.[18,35] It can be postulated that the Table 5. Factors associated with congenital CMV in multivariate model B. CMV Variables OR (95% CI) P-value P-value 25(OH)D (ng/mL) 0.10 0.46 � 32 1.00 23–31 4.88 (0.99–24.1) 0.05 <23 1.87 (0.27–12.9) 0.53 Plasma HIV-1 RNA (copies/mL) <400 1.00 400+ 0.42 (0.06–3.04) 0.39 CD4 Cell Count (copies/mL) 200+ 1.00 <200 3.08 (0.53–18.0) 0.21 Maternal age 0.90 (0.82–0.98) 0.02 P-value for trend GEE logistic regression model Abbreviations: CI, confidence interval; OR, odds ratio; 25(OH)D, 25-hydroxyvitamin D https://doi.org/10.1371/journal.pone.0228900.t005 PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 10 / 15 Low vitamin D linked to congenital CMV Table 6. Factors associated with peri/postnatal CMV in multivariate model. CMV Variables OR (95% CI) P-value P-value 1,25(OH)D (pg/mL) 0.0003 0.0008 � 116 1.00 75–115 1.60 (0.34–7.50) 0.55 � 74 9.84 (2.63–36.8) 0.0007 Plasma HIV-1 RNA (copies/mL) <400 1.00 400+ 2.18 (0.65–7.32) 0.21 CD4 Cell Count (copies/mL) 200+ 1.00 <200 1.25 (0.57–2.76) 0.58 ART Regimen 0.05 Non-HAART 1.00 HAART no PI 0.83 (0.10–7.25) 0.87 HAART with PI 4.21 (0.75–23.8) 0.10 Mode of delivery C-section 1.00 Vaginal 3.03 (1.08–8.50) 0.04 P-value for trend GEE logistic regression model Abbreviations: ART, antiretroviral therapy; CI, confidence interval; HAART, highly active antiretroviral therapy; OR, odds ratio; PI, protease inhibitor; 1,25(OH)D2, 1,25-dihydroxyvitamin D; 25(OH)D, 25-hydroxyvitamin D https://doi.org/10.1371/journal.pone.0228900.t006 additional 1,25(OH)D available to the developing fetus is in excess of the amount required for fetal skeletal development and instead may be essential for fetal and placental immunologic and antimicrobial functions. Our findings support this notion suggesting that the increased 1,25(OH)D available during pregnancy could aid immune functions important in blocking transplacental CMV transmission and possibly decrease maternal CMV shedding, thereby reducing congenital and peri/postnatal CMV infections. A prior study of untreated HIV infected pregnant women in Tanzania demonstrated increased mother-to-child-transmission of HIV in women with 25(OH)D levels <32 ng/ml. [24] In the current study, we found only weak evidence that lower levels of 25(OH)D, the main circulating form of vitamin D, may be associated with congenital CMV transmission. There was no trend as only women with levels in the middle tertile appeared to have increased CMV transmission compared to those with the highest levels, and there was no difference when comparing those with the lowest and highest levels, nor when the cut point for vitamin D sufficiency of 32 ng/ml was used for analysis. A randomized controlled trial of vitamin D supplementation in HIV negative pregnant women by Hollis, et al, demonstrated that a serum 25(OH)D level of 40 ng/ml was needed in order to achieve the supraphysiological increase in the bioactive form of vitamin D, calcitriol (1,25(OH)D ), during normal pregnancy.[25] It is possible a larger sample size is needed to similarly find a threshold serum value of 25(OH)D required to overcome any problems with placental dysfunction, supporting the necessary boost in calcitriol, and thereby aiding the important immunologic protective functions of the placenta. This study had several limitations. This is a retrospective study and the cohort was created, in part, based on availability of stored samples. This may have created inadvertent bias in PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 11 / 15 Low vitamin D linked to congenital CMV cohort selection. The women in this cohort were all considered seropositive for CMV based on maternal IgG, or early infant IgG in a few cases. Therefore, it can be assumed that the trans- missions during the congenital period were all non-primary infections due to reactivation of latent maternal CMV, or theoretically, reinfection with a different strain. Further study is needed to determine if vitamin D plays a role in low-seroprevalence populations as well, where higher rates of maternal primary CMV infection are more likely. The retrospective study design also limited the ability to do additional or confirmatory CMV PCR testing on infant urine, saliva or blood, due to the lack of availability of stored specimens. The results of this study were based mainly on culture results, as only 55 of the 340 infants had blood PCR test results, and there were no PCR results for urine or saliva. CMV cultures are less sensitive than PCR for detecting CMV and therefore the actual prevalence of congenital and peri/postnatal CMV infections may be underrepresented. More work is needed to demonstrate if the associa- tion between calcitriol, the active form of vitamin D, and congenital and early postnatal trans- mission of CMV found in this population of HIV-infected women is true in HIV uninfected women as well. Additionally, this study did not evaluate VDR expression nor the multitude of polymorphisms associated with altered VDR expression and function. It is unclear how the VDR and therefore the downstream effects of vitamin D may be impacted by CMV infection itself. In fact, a recent in vitro study demonstrated that the presence of CMV inhibited the expression of vitamin D receptors in fibroblasts.[36] Further study is needed to clarify the interplay between vitamin D and CMV infection and how this relationship may be important in protecting the developing fetus from potential infection. Although our study demonstrates an association between lower calcitriol (1,25(OH)D ) lev- els and increased CMV transmission congenitally and in the early postnatal period, we cannot comment on causation. It is unknown if the CMV and/or HIV infections deplete vitamin D as it is used in high demand in conjunction with fighting these infections, or if low maternal vita- min D leads to increased susceptibility to infection and/or increased or prolonged viral shed- ding. HIV and CMV infections are both known to cause significant placental damage and dysfunction and this could impact the placental production of calcitriol (1,25(OH)D ), poten- tially contributing to the lower levels seen in this study.[37–39] Additionally, the presence of pathogens may affect local calcitriol production. In vitro, the presence of HIV and LPS impacts the production and breakdown of calcitriol by affecting CYP27B1 and CYP24A1 gene expres- sion in monocytes.[40] Future, prospective studies are needed in HIV positive and negative populations to further clarify these complex relationships. If found to be causal and protective in prospective studies, targeted vitamin D supplementation, with the goal of supporting the necessary rise in calcitriol during pregnancy, could represent a safe and inexpensive tool in preventing CMV transmis- sion from mother to infant. Supporting information S1 Table. Number of positive CMV tests by test type for all CMV+ infants. (DOCX) S2 Table. 25-Hydroxyvitamin D sufficiency by infant transmission category. (DOCX) Acknowledgments The authors would like to acknowledge the MCA Laboratory staff for their assistance with this study. PLOS ONE | https://doi.org/10.1371/journal.pone.0228900 February 13, 2020 12 / 15 Low vitamin D linked to congenital CMV Author Contributions Conceptualization: Allison Bearden, Kristi Van Winden. Data curation: Allison Bearden, Toni Frederick, Naoko Kono, Lorayne Barton. Formal analysis: Toni Frederick, Naoko Kono. Funding acquisition: Allison Bearden, Kristi Van Winden. Investigation: Allison Bearden, Kristi Van Winden, Raj Pandian. Methodology: Allison Bearden, Kristi Van Winden, Toni Frederick, Naoko Kono, Eva Oper- skalski, Andrea Kovacs. Project administration: Allison Bearden. Resources: Andrea Kovacs. Supervision: Allison Bearden, Alice Stek, Andrea Kovacs. Writing – original draft: Allison Bearden. 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