Vitamin B12 (Cobalamin, Cbl) and folate are essential micronutrients that play major roles in important biochemical pathways in the body such as DNA synthesis and neuronal cell myelination . Deficiencies in these micronutrients are common in HIV-infected adults and are associated with a host of neurological and hematological abnormalities including, but not limited to, peripheral neuropathy, aberrant hematopoesis, low CD4 lymphocyte counts, and, especially concerning, neurocognitive dysfunction [1–4]. As many of these abnormalities can be induced by HIV-infection alone, deficiencies in Cbl and folate may often be overlooked. Studies in HIV-infected adults have demonstrated that the prevalence of vitamin B12 and folate deficiency ranges from 10–39% and 15%, respectively [4–6]. In longitudinal studies of HIV-infected adults, low serum B12 concentrations have been found to be associated with a faster progression to AIDS and a faster decline in CD4+ cell count compared with HIV-infected adults with serum B12 levels within the normal range . However, many of these studies need to be interpreted with caution as they were conducted during the ‘pre-HAART’ period and may not accurately reflect the status of micronutrient deficiencies in the current era of potent antiretroviral therapy. In addition, expanding knowledge about the role of micronutrients in disease has fostered an explosion in the use of multivitamin preparations and other nutritional supplements in the HIV clinic.
Studies evaluating the prevalence of vitamin B12 and folate deficiencies in HIV-infected children are scarce. There are no studies reporting the prevalence of these micronutrient deficiencies in HIV-infected children in the United States. We addressed this question and report the prevalence of serum vitamin B12 and folate deficiencies in a large cohort of US children with vertically acquired HIV infection. We also examined certain patient factors and laboratory tests for associations with these micronutrient deficiencies.
Patients and methods
We conducted a retrospective, cross-sectional chart review of 103 HIV-infected children and adolescents followed at Jacobi Medical Center's pediatric HIV clinic during 2001–2002. The clinic is located in an inner city hospital in the Bronx, New York, and caters to a large, primarily perinatally HIV-infected population. Medical charts were reviewed, and clinic visits 2 weeks before or after the measured vitamin B12 and folate level were examined. Data collected included demographics, serum folate and vitamin B12 levels, hematological parameters, concurrent CD4%, HIV-1 VL, Centers for Disease Control (CDC) disease category , antiretroviral regimen and other medications. Written informed consent was obtained from each participant in the study and the protocol was approved by the Investigational Review Board of Jacobi Medical Center and the Albert Einstein College of Medicine.
Serum B12 levels were measured using an Advia Centaur (Siemens Healthcare Diagnostics, Tarrytown, New York, USA) VB12 competitive immunoassay with an assay range of 26–2000 ng/l and a combined intraassay and interassay coefficient of variance (CV) of 3.9–5.9% for serum B12 at 207 −1344 ng/l. Serum folate levels were determined using the same immunoassay platform with an assay range of 0.35 – 24 μg/l and a combined intraassay and interassay CV of 7.6–8.9% for serum folate at 5.30–14.95 μg/l. Although reports in the literature differ regarding the value below which accurately defines vitamin B12 deficiency, most experts agree that serum Cbl levels less than 200 ng/l are considered deficient, whereas values between 200–300 ng/l are considered borderline . For serum folate, levels less than 2 μg/l are considered deficient, values between 3–4 μg/l are classified as borderline, and levels less than 4 μg/l effectively rule out deficiency. Although intracellular RBC folate concentrations more precisely reflect tissue folate stores, cost and availability precluded use of this assay.
The two sample T-test and the Mann–Whitney U test were used to analyze normally distributed and nonparametric variables, respectively. The degree of association between two continuous variables was assessed using the correlation coefficient. The association between elevated vitamin B12 levels and each of the potential categorical risk factors was estimated using odds ratios (OR). Parsimonious logistic regression models were fitted using P < 0.05 as the criterion for inclusion following model-building strategies suggested by Hosmer and Lemeshow . Variables that were statistically significant in the model were retained; other variables were excluded unless there was evidence of confounding (≥20% change in the parameter estimate). Models were tested for lack of fit using the Hosmer and Lemeshow goodness-of-fit test statistic and the area under ROC curve. All statistical analyses were performed using Stata software, version 9.2 (StataCorp, College Station, Texas, USA). P values were two-tailed, with an α of 0.05 considered statistically significant for all analyses.
Charts were reviewed for 103 patients followed in the HIV clinic during 2001–2002. One patient had missing data on vitamin B12 levels; hence 102 children were included in the logistic regression analysis. Demographic and laboratory features are given in Table 1. More than half the children were African–American, and only one was Caucasian. None of the children were taking multivitamin supplements during the study or up to 6 months prior; and none exhibited any clinical evidence of significant gastrointestinal malabsorption. Sixteen patients were taking cotrimoxazole (TMP-SMX) prophylaxis. Most individuals had none to moderate symptoms of HIV infection, whereas 17% (n = 18) had AIDS. All except five patients were receiving some form of combination antiretroviral therapy (ART) containing two or more medications. Adherence to these regimens was variable.
None of the children had deficiencies in vitamin B12 or serum folate. Children with elevated B12 were significantly more likely to be younger and to have higher mean serum folate levels compared with children with normal serum B12 Table 2. Serum B12 demonstrated a statistically significant positive correlation with CD4 cell count (r = 0.24; P = 0.016), white blood cell count (r = 0.28; P = 0.0047) and serum-folate levels (r = 0.34; P = 0.0005).
Median folate levels in our patients were similar to levels in healthy US children as reported by the Fifth National Health and Nutrition Examination Survey (NHANES), 2001–2002: 16.9 vs. 17 μg/l in children 4–11 years of age; and 13 vs.12.6 μg/l in children 12–19 years old. To our surprise, median serum vitamin B12 levels in our patients were higher compared with healthy US children during the same period: 1028 vs. 714 ng/l in children 4–11 years of age; and 836 vs. 515 ng/l in children 12 to 19 years of age.
On multivariate analyses, elevated levels of vitamin B12 were independently associated with elevated serum folate (OR 3.2; 95% CI 1.21, 8.37), current nonnucleoside reverse transcriptase inhibitors (NNRTI) exposure (OR 0.38; 95% CI 0.13, 1.03), and female sex (OR 0.67; 95% CI 0.25, 1.77).
This is the first report examining the prevalence of vitamin B12 and folate deficiencies in an outpatient HIV-infected pediatric cohort in the United States. None of these vertically infected children demonstrated evidence of folate or vitamin B12 deficiency, despite frequent exposure to folate antagonists like TMP-SMX, lack of multivitamin supplement use, and the possible presence of clinically occult atrophic gastritis and gastrointestinal malabsorption commonly associated with chronic-HIV infection. In this study, children with elevated serum levels of vitamin B12 were three times more likely to have elevated serum folate levels compared with children with normal B12 levels; in contrast, the use of NNRTIs and female sex were more likely to be associated with B12 levels within the normal range.
There are limited data on the prevalence of deficiencies of these essential micronutrients in HIV-infected children. Prevalence studies in HIV-infected children from Spain and Africa demonstrate that vitamin B12 and folate deficiencies are reasonably common in HAART-treated and untreated children. Vilaseca et al.  reported a 41% prevalence of folate deficiency in a cohort of HIV-infected children between 1 and 15 years of age with mean folate levels being significantly lower than levels in healthy controls. Eley et al.  reported a 5% prevalence of vitamin B12 deficiency in HIV-infected children in Cape Town, Africa.
Decreased levels of serum B12 have been associated with multiple hematological and immunological abnormalities, including: anemia, neutropenia, leukopenia, low CD4+ lymphocyte count, and abnormal activity of natural killer cells [4,13]. Although Cbl deficiency is still common amongst the current population of healthier HIV-infected adults with liberal dietary supplement use, the clinical impact of these deficiencies remain speculative. Longitudinal studies in HIV-infected patients have demonstrated that low-serum B12 levels are associated with an almost two-fold increased risk of progression to AIDS compared with patients with normal B12 levels . This association remains significant even after adjusting for markers of disease severity, suggesting that the association is causal. The mechanism, however, remains incompletely understood. In our population, serum B12 was directly correlated with CD4 lymphocyte count, WBC count and serum folate levels, suggesting that higher levels of these parameters may serve as surrogate markers for HIV-disease activity in these children.
Folate deficiency is associated with increased levels of plasma homocystine, which is a risk factor for the development of atherosclerosis and thrombosis. Hyperhomocysteinemia has been reported in adults and children with HIV-infection and is directly correlated with folate deficiency [11,14]. It is reassuring to note that the median folate levels in our population were comparable to age-matched healthy children in the United States . This suggests that routine folate fortification of foods in the United States may be playing an important role in averting deficiency of this important micronutrient in HIV-infected children in the United States. In contrast, median vitamin B12 levels were higher in our population compared with healthy US children during the 2001–2002 NHANES survey . As HIV infection routinely involves the gastrointestinal tract and can lead to vitamin B12 malabsorption by various mechanisms including decreased gastric secretion of intrinsic factor, chronic intestinal inflammation and villous atrophy [3,4,16]; our finding of elevated vitamin B12 levels in these patients is highly unexpected, and the mechanism remains unclear. Chronic immune activation, a hallmark of HIV infection, might negate the impact of these gastrointestinal perturbations by favorably altering the distribution, storage and metabolism of vitamin B12.
In summary, elevated levels of vitamin B12 are independently associated with ART regimens that do not include NNRTIs. Hence, the influence of NNRTIs on the metabolism of vitamin B12 should be explored. In addition, routine screening and supplementation with vitamin B12 and folate is not indicated without clinical or laboratory evidence of micronutrient deficiency in HIV-infected children in the United States.
There are no conflicts of interests.
Authors' role in this study: Z.A.M. worked on study design, data collection, statistical analysis and manuscript writing. J.A. did the work of study conception and manuscript writing. J.S. performed the job of data collection and manuscript writing. M.R. worked on study design, data collection and manuscript writing.
1. Hall CA. Function of vitamin B12 in the central nervous system as revealed by congenital defects. Am J Hematol 1990; 34:121–127.
2. Di Rocco A, Bottiglieri T, Werner P, Geraci A, Simpson D, Godbold J, Morgello S. Abnormal cobalamin-dependent transmethylation in AIDS-associated myelopathy. Neurology 2002; 58:730–735.
3. Remacha AF, Cadafalch J. Cobalamin deficiency in patients infected with the human immunodeficiency virus. Semin Hematol 1999; 36:75–87.
4. Remacha AF, Riera A, Cadafalch J, Gimferrer E. Vitamin B-12 abnormalities in HIV-infected patients. Eur J Haematol 1991; 47:60–64.
5. Burkes RL, Cohen H, Krailo M, Sinow RM, Carmel R. Low serum cobalamin levels occur frequently in the acquired immune deficiency syndrome and related disorders. Eur J Haematol 1987; 38:141–147.
6. Ehrenpreis ED, Carlson SJ, Boorstein HL, Craig RM. Malabsorption and deficiency of vitamin B12 in HIV-infected patients with chronic diarrhea. Dig Dis Sci 1994; 39:2159–2162.
7. Tang AM, Graham NM, Chandra RK, Saah AJ. Low serum vitamin B-12 concentrations are associated with faster human immunodeficiency virus type 1 (HIV-1) disease progression. J Nutr 1997; 127:345–351.
8. 1993 revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. MMWR Recomm Rep
9. Snow CF. Laboratory diagnosis of vitamin B12 and folate deficiency: a guide for the primary care physician. Arch Intern Med 1999; 159:1289–1298.
10. Hosmer DW, Lemeshow S. Model-building strategies and methods for logistic regression
. In: Applied logistic regression
. New York: Wiley-Interscience Publication; 2000, pp.
11. Vilaseca MA, Sierra C, Colome C, Artuch R, Valls C, Munoz-Almagro C, et al
. Hyperhomocysteinaemia and folate deficiency in human immunodeficiency virus-infected children. Eur J Clin Invest 2001; 31:992–998.
12. Eley BS, Sive AA, Abelse L, Kossew G, Cooper M, Hussey GD. Growth and micronutrient disturbances in stable, HIV-infected children in Cape Town. Ann Trop Paediatr 2002; 22:19–23.
13. Mantero-Atienza E, Baum MK, Morgan R, Wilkie F, Shor-Posner G, Fletcher MA, et al
. Vitamin B12 in early human immunodeficiency virus-1 infection. Arch Intern Med 1991; 151:1019–1020.
14. Uccelli MC, Torti C, Lapadula G, Labate L, Cologni G, Tirelli V, et al
. Influence of folate serum concentration on plasma homocysteine levels in HIV-positive patients exposed to protease inhibitors undergoing HAART. Ann Nutr Metab 2006; 50:247–252.
15. Pfeiffer CM, Johnson CL, Jain RB, Yetley EA, Picciano MF, Rader JI, et al
. Trends in blood folate and vitamin B-12 concentrations in the United States, 1988 2004. Am J Clin Nutr 2007; 86:718–727.
16. Lake-Bakaar G, Elsakr M, Hagag N, Lyubsky S, Ahuja J, Craddock B, Steigbigel RT. Changes in parietal cell structure and function in HIV disease. Dig Dis Sci 1996; 41:1398–1408.