Guidelines for the Use of Antiretroviral Agents in Pediatric HIV Infection
The information in the brief version is excerpted directly from the full-text guidelines. The brief version is a compilation of the tables and boxed recommendations.
Nucleoside and Nucleotide Analogue Reverse Transcriptase Inhibitors (NRTIs)
Last Updated: May 22, 2018; Last Reviewed: May 22, 2018
|Tenofovir Disoproxil Fumarate (TDF, Viread)
For additional information see Drugs@FDA: http://www.accessdata.fda.gov/scripts/cder/daf/
|Oral Powder: 40 mg per 1 g of oral powder (1 level scoop, measured with supplied dosing scoop = 1 g oral powder)
Tablets: 150 mg, 200 mg, 250 mg, and 300 mg
Fixed-Dose Combination Tablets:
|Dosing Recommendations||Selected Adverse Events|
|Neonate and Infant Dose:
TDF Tablet Dosing Table for Patients Aged ≥2 Years and Weighing ≥17 kg
Adolescent (Weighing ≥35 kg)a and Adult Dose:
[Truvada] Emtricitabine plus TDF (FTC/TDF)
Truvada Pediatric Dosing Table
Adolescent (Weighing ≥35 kg) and Adult Dose:
Adolescent (Weighing ≥40 kg) and Adult Dose:
Adolescent (Weighing ≥35 kg) and Adult Dose:
Adolescent (Weighing >35 kg with a Sexual Maturity Rating of 4 or 5) and Adult Dose:
Pediatric (Weighing ≥35 kg) and Adult Dose:
|a See text for concerns about decreased bone mineral density, especially in prepubertal patients and those in early puberty (Sexual Maturity Rating 1 and 2, previously called Tanner staging).|
- Tenofovir disoproxil fumarate (TDF) is a substrate of the adenosine triphosphate-dependent transporters P-glycoprotein (P-gp) and breast cancer resistance protein. When TDF is co-administered with inhibitors of these transporters, an increase in TDF absorption may be observed, with the potential for enhanced TDF toxicity.1
- Renal elimination: Drugs that decrease renal function or compete for active tubular secretion could reduce clearance of plasma tenofovir (TFV).
- Other nucleoside reverse transcriptase inhibitors (NRTIs): Didanosine serum concentrations increase when the drug is co-administered with TDF, and this combination should not be used because of increase in didanosine toxicity.
- Protease inhibitors: Because TDF decreases atazanavir plasma concentrations, atazanavir without ritonavir should not be co-administered with TDF. In addition, the combination of atazanavir and lopinavir/ritonavir increases plasma tenofovir concentrations and potentiates TDF-associated toxicity.2
- Use of Stribild: If using Stribild, please see the Elvitegravir section of the drug appendix for additional information
- More common: Nausea, diarrhea, vomiting, and flatulence.
- Less common (more severe): TDF caused bone toxicity (osteomalacia and reduced bone mineral density [BMD]) in animals when given in high doses. Decreases in BMD have been reported in both adults and children taking TDF. Renal toxicity, including increased serum creatinine, glycosuria, proteinuria, phosphaturia, and/or calciuria and decreased serum phosphate, has been observed. Patients at increased risk of renal glomerular or tubular dysfunction should be closely monitored. Cases of lactic acidosis and severe hepatomegaly with steatosis, including fatal cases, have been reported.
TDF is Food and Drug Administration (FDA)-approved for use in children aged ≥2 years when used as a component of antiretroviral therapy (ART).
TDF has antiviral activity and efficacy against hepatitis B virus (HBV) and is FDA-approved for HBV treatment in children aged ≥12 years. The use of TDF to treat HIV/HBV coinfection is reviewed in the Pediatric Opportunistic Infection Guidelines.
Efficacy in Clinical Trials in Adults Compared to Children and Adolescents
The standard adult dose approved by the FDA for adults and children aged ≥12 years and weighing ≥35 kg is TDF 300 mg once daily. For children aged 2 years to 12 years, the FDA-approved dose is TDF 8 mg/kg/dose administered once daily, which closely approximates the dose of TDF 208 mg/m2/dose used in early studies in children.3
In adults, the recommended TDF dose is highly effective. In comparative clinical trials in adults, TDF administered with lamivudine or emtricitabine as a dual-NRTI backbone in combination with efavirenz had better viral efficacy than zidovudine used with lamivudine and efavirenz.4-6 TDF administered with emtricitabine has been compared to abacavir administered with lamivudine in several adult studies and metaanalyses, with variable results.7-11
In children, the published efficacy data for TDF are mixed, but potency equal to that in adults is seen in pediatric patients aged 3 years to 18 years with susceptible virus. In children aged 2 years to <12 years, TDF 8 mg/kg/dose once daily was noninferior to twice-daily zidovudine- or stavudine-containing ART over 48 weeks of randomized treatment.12,13 Virologic success is lower in treatment-experienced patients with extensive drug resistance.14-16
Relationship of Drug Exposure to Virologic Response
Virologic suppression is most closely related to intracellular tenofovir diphosphate (TFV-DP) concentrations, and for TDF, intracellular TFV-DP is linked to plasma TFV concentration.17 A modeling study suggests that children and adolescents treated with TDF may have higher intracellular TFV-DP concentrations than adults,18 even though plasma TFV concentrations are lower in children and adolescents, because renal clearance of TFV is higher in children than in adults.3,19,20
The taste-masked granules that make up the TDF oral powder give the vehicle (e.g., applesauce, yogurt) a gritty consistency. Once mixed with a vehicle, TDF should be administered promptly because its taste becomes bitter if it is allowed to sit too long.
TDF administration is associated with decreased BMD in both adults21,22 and children.13,23-25 When treated with TDF, younger children with Sexual Maturity Ratings (SMRs; previously Tanner Stages) 1 and 2 may be at higher risk of decreased BMD than children with more advanced pubertal development (i.e., SMR ≥3).19 Discontinuation of TDF results in partial or complete recovery of BMD.23
In the industry-sponsored study that led to FDA approval of TDF in adolescents aged ≥12 years and weighing ≥35 kg, six of 33 participants (18%) in the TDF arm experienced a >4% decline in absolute lumbar spine BMD in 48 weeks, while only one of 33 participants (3%) in the placebo arm experienced this decline.14
TDF administration disrupts vitamin D metabolism,26,27 and the decrease in BMD associated with TDF initiation was attenuated in adults with co-administration of high doses of vitamin D3 (4000 International Units [IU] daily) and calcium carbonate (1000 mg daily) for the first 48 weeks of TDF treatment.28 During chronic TDF administration, youth with HIV who received vitamin D3 supplements (50,000 IU once monthly) had decreased serum parathyroid hormone29 and increased lumbar spine BMD.27 The serum 25-hydroxy vitamin D concentration in the group with improved BMD was 37 ng/mL. Since this improvement in lumbar spine BMD was seen in patients with and without baseline vitamin D deficiency, some practitioners recommend vitamin D supplementation in all patients treated with TDF-containing ART. Plasma concentrations of the TDF metabolite plasma TFV have been associated with TDF-related endocrine disruption and low BMD.26 Tenofovir alafenamide (TAF), which is associated with lower plasma TFV concentrations than TDF, causes less decline in BMD than TDF (see the Tenofovir Alafenamide section for more information). Consider switching from TDF to TAF in appropriate clinical settings.
Monitoring Potential Bone Toxicity
The Panel on Antiretroviral Therapy and Medical Management of Children Living with HIV (the Panel) does not recommend routine dual-energy absorptiometry (DXA) monitoring for children or adolescents treated with TDF. Given the potential for BMD loss in children treated with TDF, some experts obtain a DXA before initiation of TDF therapy and approximately 6 months after starting TDF, especially in prepubertal patients and those early in puberty (i.e., SMR 1 and 2). If DXA results are abnormal, consider referral to a subspecialist in pediatric endocrinology or a related field.
Despite the ease of use of a once-daily drug and the efficacy of TDF, the potential for BMD loss during the important period of rapid bone accrual in childhood and early adolescence is concerning and favors use of abacavir or TAF in children with SMRs 1 to 3, because children with perinatally acquired HIV are at risk for low peak bone mass.30,31
New onset or worsening of renal impairment has been reported in adults32 and children33,34 receiving TDF. In one study, renal toxicity led to discontinuation of TDF in 3.7% of children with HIV (6/159 children) who were treated with TDF.16 While TDF is clearly associated with a decline in glomerular filtration rate, the effect is generally small, and severe glomerular toxicity is rare.32,33 Irreversible renal failure is quite rare, but cases have been reported.35
The main target of TDF nephrotoxicity is the renal proximal tubule.33 Case reports highlight the infrequent but most severe manifestations of renal Fanconi syndrome, hypophosphatemia, hypocalcemia, diabetes insipidus, myalgias, bone pain, and fractures.36,37
Subclinical renal tubular damage is more common than clinically apparent renal tubular injury. Increased urinary beta-2 microglobulin was identified in 27% of children (12/44 children) treated with TDF and in 4% of children (2/48 children) not treated with TDF.38 The risks of TDF-associated proteinuria and chronic kidney disease increase with the duration of treatment.39,40 Of 89 participants aged 2 years to 12 years who received TDF in Gilead Study 352 (where participants had a median drug exposure of 104 weeks), four participants were discontinued from the study for renal tubular dysfunction, with the discontinuations occurring between 84 and 156 weeks on TDF therapy.12
Plasma TFV is the TDF metabolite most closely associated with both glomerular26,41 and proximal tubular42 toxicity. TAF, which generates lower plasma TFV concentrations than TDF, is associated with less renal toxicity than TDF (see the Tenofovir Alafenamide section).
Monitoring Potential Renal Toxicity
Because TDF has the potential to decrease creatinine clearance and cause renal tubular dysfunction, the Panel recommends measuring serum creatinine and using a urine dipstick to check protein and glucose levels prior to drug initiation. It is unclear how often creatinine and renal tubular function (urine protein and glucose) should be monitored in asymptomatic patients. Many Panel members monitor creatinine with other blood tests every 3 to 4 months and perform urinalysis every 6 to 12 months. Serum phosphate should be measured if clinically indicated; renal phosphate loss can occur in the presence of normal creatinine and the absence of proteinuria. Because nephrotoxicity increases with the duration of TDF treatment, monitoring should be continued during long-term therapy with the drug.
Because renal glomerular damage primarily increases the urine concentration of albumin, and proximal renal tubular damage increases urine concentrations of low-molecular-weight proteins like beta-2 microglobulin, dipstick urinalysis (measuring primarily urine albumin) may be a relatively insensitive marker for TDFassociated tubular damage. Measuring urine albumin and urine protein and calculating the urine albumin to urine protein ratio can be helpful in identifying the nonalbumin proteinuria that is seen in TDF-associated nephrotoxicity.43,44 While these more complex and expensive tests may be used in research settings, in clinical practice, renal tubular damage is perhaps easiest to identify by using a renal dipstick to identify normoglycemic glycosuria and proteinuria.
- Tenofovir disoproxil fumarate [package insert]. Food and Drug Administration. 2017. Available at: https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/021356s055,022577s011lbl.pdf.
- Bedimo R, Rosenblatt L, Myers J. Systematic review of renal and bone safety of the antiretroviral regimen efavirenz, emtricitabine, and tenofovir disoproxil fumarate in patients with HIV infection. HIV Clin Trials. 2016;17(6):246-266. Available at: https://www.ncbi.nlm.nih.gov/pubmed/27809711.
- Hazra R, Balis FM, Tullio AN, et al. Single-dose and steady-state pharmacokinetics of tenofovir disoproxil fumarate in human immunodeficiency virus-infected children. Antimicrob Agents Chemother. 2004;48(1):124-129. Available at: http://www.ncbi.nlm.nih.gov/pubmed/14693529.
- Arribas JR, Pozniak AL, Gallant JE, et al. Tenofovir disoproxil fumarate, emtricitabine, and efavirenz compared with zidovudine/lamivudine and efavirenz in treatment-naive patients: 144-week analysis. J Acquir Immune Defic Syndr. 2008;47(1):74-78. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17971715.
- Gallant JE, Staszewski S, Pozniak AL, et al. Efficacy and safety of tenofovir DF vs. stavudine in combination therapy in antiretroviral-naive patients: a 3-year randomized trial. JAMA. 2004;292(2):191-201. Available at: http://www.ncbi.nlm.nih.gov/pubmed/15249568.
- Gallant JE, DeJesus E, Arribas JR, et al. Tenofovir DF, emtricitabine, and efavirenz vs. zidovudine, lamivudine, and efavirenz for HIV. N Engl J Med. 2006;354(3):251-260. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16421366.
- Sax PE, Tierney C, Collier AC, et al. Abacavir-lamivudine versus tenofovir-emtricitabine for initial HIV-1 therapy. N Engl J Med. 2009;361(23):2230-2240. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19952143.
- Sax PE, Tierney C, Collier AC, et al. Abacavir/lamivudine versus tenofovir DF/emtricitabine as part of combination regimens for initial treatment of HIV: final results. J Infect Dis. 2011;204(8):1191-1201. Available at: http://www.ncbi.nlm.nih.gov/pubmed/21917892.
- Smith KY, Patel P, Fine D, et al. Randomized, double-blind, placebo-matched, multicenter trial of abacavir/lamivudine or tenofovir/emtricitabine with lopinavir/ritonavir for initial HIV treatment. AIDS. 2009;23(12):1547-1556. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19542866.
- Post FA, Moyle GJ, Stellbrink HJ, et al. Randomized comparison of renal effects, efficacy, and safety with once-daily abacavir/lamivudine versus tenofovir/emtricitabine, administered with efavirenz, in antiretroviral-naive, HIV-1-infected adults: 48-week results from the ASSERT study. J Acquir Immune Defic Syndr. 2010;55(1):49-57. Available at: http://www.ncbi.nlm.nih.gov/pubmed/20431394.
- Spaulding A, Rutherford GW, Siegfried N. Tenofovir or zidovudine in three-drug combination therapy with one nucleoside reverse transcriptase inhibitor and one non-nucleoside reverse transcriptase inhibitor for initial treatment of HIV infection in antiretroviral-naive individuals. Cochrane Database Syst Rev. 2010(10):CD008740. Available at: http://www.ncbi.nlm.nih.gov/pubmed/20927777.
- Saez-Llorens X, Castano E, Rathore M, et al. A randomized, open-label study of the safety and efficacy of switching stavudine or zidovudine to tenofovir disoproxil fumarate in HIV-1-infected children with virologic suppression. Pediatr Infect Dis J. 2015;34(4):376-382. Available at: http://www.ncbi.nlm.nih.gov/pubmed/25760565.
- Aurpibul L, Cressey TR, Sricharoenchai S, et al. Efficacy, safety and pharmacokinetics of tenofovir disoproxil fumarate in virologic-suppressed HIV-infected children using weight-band dosing. Pediatr Infect Dis J. 2015;34(4):392-397. Available at: http://www.ncbi.nlm.nih.gov/pubmed/25760566.
- Della Negra M, de Carvalho AP, de Aquino MZ, et al. A randomized study of tenofovir disoproxil fumarate in treatment-experienced HIV-1 infected adolescents. Pediatr Infect Dis J. 2012;31(5):469-473. Available at: http://www.ncbi.nlm.nih.gov/pubmed/22301477.
- Della Negra M, De Carvalho AP, De Aquino MZ, et al. Long-term efficacy and safety of tenofovir disoproxil fumarate in HIV-1-infected adolescents failing antiretroviral therapy: the final results of study GS-US-104-0321. Pediatr Infect Dis J. 2015;34(4):398-405. Available at: http://www.ncbi.nlm.nih.gov/pubmed/25599284.
- Riordan A, Judd A, Boyd K, et al. Tenofovir use in human immunodeficiency virus-1-infected children in the United kingdom and Ireland. Pediatr Infect Dis J. 2009;28(3):204-209. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19209091.
- Baheti G, Kiser JJ, Havens PL, Fletcher CV. Plasma and intracellular population pharmacokinetic analysis of tenofovir in HIV-1-infected patients. Antimicrob Agents Chemother. 2011;55(11):5294-5299. Available at: http://www.ncbi.nlm.nih.gov/pubmed/21896913.
- Baheti G, King JR, Acosta EP, Fletcher CV. Age-related differences in plasma and intracellular tenofovir concentrations in HIV-1-infected children, adolescents and adults. AIDS. 2013;27(2):221-225. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23032419.
- Hazra R, Gafni RI, Maldarelli F, et al. Tenofovir disoproxil fumarate and an optimized background regimen of antiretroviral agents as salvage therapy for pediatric HIV infection. Pediatrics. 2005;116(6):e846-854. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16291735.
- Kiser JJ, Fletcher CV, Flynn PM, et al. Pharmacokinetics of antiretroviral regimens containing tenofovir disoproxil fumarate and atazanavir-ritonavir in adolescents and young adults with human immunodeficiency virus infection. Antimicrob Agents Chemother. 2008;52(2):631-637. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18025112.
- Stellbrink HJ, Orkin C, Arribas JR, et al. Comparison of changes in bone density and turnover with abacavir-lamivudine versus tenofovir-emtricitabine in HIV-infected adults: 48-week results from the ASSERT study. Clin Infect Dis. 2010;51(8):963-972. Available at: http://www.ncbi.nlm.nih.gov/pubmed/20828304.
- McComsey GA, Kitch D, Daar ES, et al. Bone mineral density and fractures in antiretroviral-naive persons randomized to receive abacavir-lamivudine or tenofovir disoproxil fumarate-emtricitabine along with efavirenz or atazanavir-ritonavir: AIDS Clinical Trials Group A5224s, a substudy of ACTG A5202. J Infect Dis. 2011;203(12):1791-1801. Available at: http://www.ncbi.nlm.nih.gov/pubmed/21606537.
- Gafni RI, Hazra R, Reynolds JC, et al. Tenofovir disoproxil fumarate and an optimized background regimen of antiretroviral agents as salvage therapy: impact on bone mineral density in HIV-infected children. Pediatrics. 2006;118(3):e711-718. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16923923.
- Purdy JB, Gafni RI, Reynolds JC, Zeichner S, Hazra R. Decreased bone mineral density with off-label use of tenofovir in children and adolescents infected with human immunodeficiency virus. J Pediatr. 2008;152(4):582-584. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18346519.
- Aurpibul L, Puthanakit T. Review of tenofovir use in HIV-infected children. Pediatr Infect Dis J. 2015;34(4):383-391. Available at: http://www.ncbi.nlm.nih.gov/pubmed/25247583.
- Havens PL, Kiser JJ, Stephensen CB, et al. Association of higher plasma vitamin D binding protein and lower free calcitriol levels with tenofovir disoproxil fumarate use and plasma and intracellular tenofovir pharmacokinetics: cause of a functional vitamin D deficiency? Antimicrob Agents Chemother. 2013;57(11):5619-5628. Available at: http://www.ncbi.nlm.nih.gov/pubmed/24002093.
- Havens PL, Stephensen CB, Van Loan MD, et al. Vitamin D3 supplementation increases spine bone mineral density in adolescents and young adults with human immunodeficiency virus infection being treated with tenofovir disoproxil fumarate: a randomized, placebo-controlled trial. Clin Infect Dis. 2018;66(2):220-228. Available at: https://www.ncbi.nlm.nih.gov/pubmed/29020329.
- Overton ET, Chan ES, Brown TT, et al. Vitamin D and calcium attenuate bone loss with antiretroviral therapy initiation: a randomized trial. Ann Intern Med. 2015;162(12):815-824. Available at: http://www.ncbi.nlm.nih.gov/pubmed/26075752.
- Havens PL, Stephensen CB, Hazra R, et al. Vitamin D3 decreases parathyroid hormone in HIV-infected youth being treated with tenofovir: a randomized, placebo-controlled trial. Clin Infect Dis. 2012;54(7):1013-1025. Available at: http://www.ncbi.nlm.nih.gov/pubmed/22267714.
- DiMeglio LA, Wang J, Siberry GK, et al. Bone mineral density in children and adolescents with perinatal HIV infection. AIDS. 2013;27(2):211-220. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23032412.
- Yin MT, Lund E, Shah J, et al. Lower peak bone mass and abnormal trabecular and cortical microarchitecture in young men infected with HIV early in life. AIDS. 2014;28(3):345-353. Available at: http://www.ncbi.nlm.nih.gov/pubmed/24072196.
- Cooper RD, Wiebe N, Smith N, Keiser P, Naicker S, Tonelli M. Systematic review and meta-analysis: renal safety of tenofovir disoproxil fumarate in HIV-infected patients. Clin Infect Dis. 2010;51(5):496-505. Available at: http://www.ncbi.nlm.nih.gov/pubmed/20673002.
- Hall AM. Update on tenofovir toxicity in the kidney. Pediatr Nephrol. 2013;28(7):1011-1023. Available at: http://www.ncbi.nlm.nih.gov/pubmed/22878694.
- Andiman WA, Chernoff MC, Mitchell C, et al. Incidence of persistent renal dysfunction in human immunodeficiency virus-infected children: associations with the use of antiretrovirals, and other nephrotoxic medications and risk factors. Pediatr Infect Dis J. 2009;28(7):619-625. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19561425.
- Wood SM, Shah SS, Steenhoff AP, Meyers KE, Kaplan BS, Rutstein RM. Tenofovir-associated nephrotoxicity in two HIV-infected adolescent males. AIDS Patient Care STDS. 2009;23(1):1-4. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19183077.
- Hussain S, Khayat A, Tolaymat A, Rathore MH. Nephrotoxicity in a child with perinatal HIV on tenofovir, didanosine and lopinavir/ritonavir. Pediatr Nephrol. 2006;21(7):1034-1036. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16773419.
- Lucey JM, Hsu P, Ziegler JB. Tenofovir-related Fanconi's syndrome and osteomalacia in a teenager with HIV. BMJ Case Rep. 2013;2013. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23843401.
- Papaleo A, Warszawski J, Salomon R, et al. Increased beta-2 microglobulinuria in human immunodeficiency virus-1-infected children and adolescents treated with tenofovir. Pediatr Infect Dis J. 2007;26(10):949-951. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17901802.
- Soler-Palacin P, Melendo S, Noguera-Julian A, et al. Prospective study of renal function in HIV-infected pediatric patients receiving tenofovir-containing HAART regimens. AIDS. 2011;25(2):171-176. Available at: http://www.ncbi.nlm.nih.gov/pubmed/21076275.
- Purswani M, Patel K, Kopp JB, et al. Tenofovir treatment duration predicts proteinuria in a multiethnic United States Cohort of children and adolescents with perinatal HIV-1 infection. Pediatr Infect Dis J. 2013;32(5):495-500. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23249917.
- Poizot-Martin I, Solas C, Allemand J, et al. Renal impairment in patients receiving a tenofovir-cART regimen: impact of tenofovir trough concentration. J Acquir Immune Defic Syndr. 2013;62(4):375-380. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23196828.
- Rodriguez-Novoa S, Labarga P, D'Avolio A, et al. Impairment in kidney tubular function in patients receiving tenofovir is associated with higher tenofovir plasma concentrations. AIDS. 2010;24(7):1064-1066. Available at: http://www.ncbi.nlm.nih.gov/pubmed/20299966.
- Sise ME, Hirsch JS, Canetta PA, Herlitz L, Mohan S. Nonalbumin proteinuria predominates in biopsy-proven tenofovir nephrotoxicity. AIDS. 2015;29(8):941-946. Available at: http://www.ncbi.nlm.nih.gov/pubmed/25784440.
- Samarawickrama A, Cai M, Smith ER, et al. Simultaneous measurement of urinary albumin and total protein may facilitate decision-making in HIV-infected patients with proteinuria. HIV Med. 2012;13(9):526-532. Available at: http://www.ncbi.nlm.nih.gov/pubmed/22413854.