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Protocol Team Members (versions 1 and 2 of PACTG-219): Mary Culnane MS CRNP, Elizabeth Hawkins, Lynne Mofenson MD, Yvonne J Bryson, MD, Edward M Connor MD, Lawrence D'Angelo MD MPH, Mark Mintz MD, Karen J O'Donnell PhD, Margaret Oxtoby, MD, Andrea Rubin Hale RN MPH, Richard D Gelber PhD, Steven Gortmaker PhD, William Lenderking PhD, Lynn Marrow, Christina Joy RN MSM, Colleen Clark MPH, Bethann Cunningham MS, Rhoda Sperling MD, Gwendolyn B Scott MD, Courtney Fletcher PharmD, Blake Caldwell MD, Dianne Donovan,
Protocol Team Members (versions 3 and 4 of PACTG-219C): Elizabeth Smith MD, Anne Fresia, Gregory Ciupak, Michelle Eagle PA, Dorothy R Smith MS CPNP, Paul Palumbo MD, John Sleasman MD, James Connor MD, Michael Hughes PhD, Rebecca Oyomopita MSc, George Johnson MD, Andrew Wiznia MD, Nancy Hutton MD, Andrea Kovacs MD, Mary Sawyer MD, Martin Anderson MD, Audrey Rogers PhD MPH, William Borkowsky MD, Jane Lindsey ScD, Jack Moye MD, Myron Levin MD, Marilyn Crain MD MPH, Paul Britto MS, Ruth Toumala MD, Joseph Cervia MD, Eileen Monagham, Kenneth Dominguez MD, Melody Higgins RN MS, George Seage DSc MPH, Denise Gaughan MPH, Phil Gona PhD, William Shearer MD PhD, Lois Howland DPH MS RN, Deborah Storm PhD RN, Kathleen Malee PhD, Wendy Mitchell MD, Carol Gore, Eve Powell, Michelle McConnell MD, Newana Beatty, Susan Brogly PhD, Jennifer Bryant CRA, Miriam Chernoff PhD, Barbara Heckman BS, Dawn English, Edward Handelsman MD, Patrick Jean-Philippe MD, Kathleen Kaiser, Joyce Kraimer MS, Linda Millar, Shirley Traite MSW, Paige Williams PhD, Elizabeth Woods MD MPH, Carol Worrell MD.
Mitochondrial dysfunction (MD) has been associated with both HIV infection and exposure to antiretroviral therapies. MD has not been widely studied in HIV-infected children.
Children with perinatal HIV infection enrolled in a prospective cohort, Pediatric AIDS Clinical Trials Group 219/ 219C, between 1993 and 2004 were included. Two clinical case definitions of MD, the Enquête Périnatale Française criteria and the Mitochondrial Disease Classification, were used to classify signs and symptoms consistent with possible MD. Adjusted odds ratios of the associations between single and dual nucleoside reverse transcriptase inhibitor (NRTI) use and possible MD were estimated using logistic regression.
Overall, 982/2931 children (33.5%) met one or both case definitions of possible MD. Mortality was highest among the 96 children meeting both case definitions (20%). After adjusting for confounders, children using d4T regardless of other exposures (OR 3.44, 95% CI 1.91, 6.20) or d4T/ddI combination therapy (OR 2.23, 95% CI 1.19, 4.21) had a higher risk of possible MD. 3TC and 3TC/d4T exposures also were associated with increased MD.
NRTIs, especially d4T and 3TC, were associated with possible MD in children with perinatal HIV. Further studies are warranted to elucidate potential mechanisms of NRTI toxicities.
Mitochondrial dysfunction (MD) has been reported in children and adults infected with HIV. Most studies attribute this finding to antiretroviral medications (ARVs), specifically nucleoside reverse transcriptase inhibitors (NRTIs)[1-4]. However MD has also been reported in HIV-infected individuals naïve to ARVs [5, 6]. Before an effective prevention or treatment strategy can be devised, it is important to determine the frequency of MD in HIV-infected children and to identify factors associated with its development [6-8].
Studies of the etiology of MD in HIV-infected individuals have been hampered by diagnostic difficulties. The constellation of clinical findings in some individuals with HIV infection is similar to that of confirmed primary mitochondrial disorders [1, 9]. Clinical signs and symptoms of MD may not be recognized in advanced HIV disease with marked elevations of HIV viral load treated with multiple antiretroviral medications. Expression of MD may be related to host mitochondrial haplotypes or other genetic characteristics [10, 11], defects in intergenomic communication such as mitochondrial DNA depletion syndromes , environmental influences including HIV infection [13, 14], and specific ARVs [14-17]. Mitochondrial toxicity due to NRTI use in HIV-infected adults has been reported with zidovudine (ZDV), lamivudine (3TC), and stavudine (d4T) [4, 17-23], and in children with didanosine (ddI) exposure [24, 25].
The main objectives of this study were to estimate the frequency of clinically defined MD in children with perinatal HIV infection and to study its association with exposure to specific ARVs, specifically NRTIs.
The Pediatric AIDS Clinical Trials Group (PACTG) protocol “219: Pediatric Late Outcomes Study” began May 4, 1993, as a prospective cohort study to assess the late effects of in utero and neonatal exposure to ARVs in HIV-exposed children and of ARV use in HIV-infected children. All children who had participated in PACTG perinatal or pediatric trials in the United States were eligible for enrollment into 219, which ended on September 15, 2000. This was followed by the introduction of a new protocol version, 219C, which did not require prior participation in a PACTG perinatal or treatment trial for enrollment eligibility. Sixty-six percent of the 219 participants with HIV infection enrolled into 219C, in addition to HIV-infected children not previously enrolled [26, 27]. These studies have been described elsewhere [26, 28, 29].
The study population for the present study consisted of children perinatally infected with HIV enrolled into PACTG 219 or 219C between May 4, 1993 and November 3, 2004 with at least two study visits at three-month intervals and at least six months of laboratory follow-up prior to closure of the dataset on May 3, 2005. Study sites obtained approval from their respective Institutional Review Boards for Human Research and written consent from the child's parent or guardian.
Visits occurred every three months until protocol completion, withdrawal, loss-to-follow-up, or death. At enrollment and follow-up in 219/219C, clinical diagnoses and diagnostic test results were recorded and physical examinations and neurologic evaluations were performed. Laboratory testing (chemistry and hematology), echocardiograms, neuropsychological testing (Bayley Scales of Infant Development, Wechsler tests), and audiometric data were collected according to the protocol schedule or as clinically required. All signs and symptoms and blood chemistry and hematology data were assigned a toxicity grade, and together with diagnoses, were coded utilizing MedDRA®, the Medical Dictionary for Regulatory Activities by professional medical coders. MedDRA® is an international terminology used in all phases of drug development for data entry, retrieval and analysis. In the context of this study, MedDRA® was used to code reported adverse events and then utilized to retrieve those adverse events according to case definitions [30, 31].
Children were considered to have possible MD if they met criteria for at least one of two case definitions [32, 33]. The Enquête Périnatale Française (EPF) criteria have been used primarily to evaluate possible signs of MD in HIV-uninfected infants exposed both to maternal HIV infection and ART in utero, but who proved to be free of HIV infection [28, 32]. To meet this definition, children had at least one major condition (neurologic or other organ system) at any visit or two minor conditions reported on at least two visits .
The Mitochondrial Disease Criteria (MDC) was developed as a clinical tool utilizing a point system to classify children in the general pediatric population whose constellation of signs and symptoms is unlikely (0-1 point), possibly (2-4 points), probably (5-7 points), or definitely (8-12 points) related to mitochondrial respiratory chain disorders . Points are assigned for 40 diagnoses according to the primary clinical presentation: muscular (maximum of 2 points), central nervous system (maximum 2 points), or multisystem (maximum of 3 points), with additional points added for subsequent clinical conditions, to a maximum of 4 points for clinical findings. For more than 4 points to be accumulated, additional specified testing for mitochondrial disorders, including metabolic, laboratory, and tissue abnormalities is required (http://www.neurology.org/cgi/content/full/59/9/1402/DC1/1). Children with ≥ 2 points may then be evaluated further with invasive tests, including muscle biopsy for histology and metabolic testing for further point accumulation to definitively establish a respiratory chain defect. Since protocols 219/219C did not require biochemical or histological examination of tissue to diagnose mitochondrial disorders, 4 points was the maximum possible score. Study participants with ≥ 2 points (the minimum point threshold for reaching the category of possible MD within the MDC) were considered to be cases of possible MD. Both clinical and laboratory criteria for possible MD required development of definitions for persistence (three months or longer) and severity (≥ Grade 2) for each relevant condition. The 1994 National Institutes of Health Division of Allergy and Infectious Diseases Toxicity Grading Tables were used in this study.
Children with clinical and laboratory abnormalities meeting criteria for EPF and MDC case definitions separately were identified. Diagnoses or laboratory values triggering case definition conditions were systematically reviewed. The primary function of the review was to eliminate diagnoses, terms, and laboratory values where an alternative explanation to MD was plausible (e.g. neurosensory hearing loss in a child with confirmed cytomegalovirus infection). Clinicians were blinded to ARV treatment and reviewed only information deemed relevant to a particular criterion in order to maintain reviewer objectivity and study integrity.
For the MDC criteria, the date at which the participant reached his/her maximum MDC score was considered the date of MD diagnosis. For EPF criteria, the date of MD diagnosis was the date of the earliest event meeting the case criteria. Prevalent MD cases were those whose event date occurred at or prior to study enrollment.
ARV use was classified by individual NRTI and specific NRTI combinations. Single NRTI agents commonly used as a component of ARV therapy were considered (3TC, ZDV, d4T, and ddI) as well as common NRTI combinations (3TC/d4T, ZDV/ddI, ZDV/3TC, and d4T/ddI). ARV use in the year prior to the presentation of clinical signs of possible MD or in the year prior to the end of study follow-up if no MD event occurred was included.
Prevalence and incidence of MD by EPF and MDC definitions were estimated. For concordance of incident events, cases either positive or negative for both EPF and MDC criteria were evaluated. Cases meeting either case definition prior to study entry were considered prevalent and excluded from the incidence analysis.
Logistic regression was used to estimate the association between ART use and incident MD, while controlling for potential confounders identified a priori: gender, race and ethnicity, age at study entry, neonatal ZDV prophylaxis, maternal ART exposure (prenatal and intrapartum), study entry CDC clinical category , time-adjusted AUC (area under the curve) HIV RNA viral load (VL), and most recent CD4% prior to the year of ARV use. CDC clinical category, VL and CD4% were included since HIV disease severity may relate to both ARV use and possible MD. The standard of care for HIV-infected children evolved during the study period; thus the number of years prior to (negative) or after (positive) 1995 prior to the event or end of follow-up were also included in the logistical analysis.
The association between MD and ARVs was estimated for each of four single NRTIs and four NRTI combinations. In each analysis the referent group comprised children who were not receiving the ARV being studied during the year prior to event or censor time, although they may have received other ARVs during this exposure year. Sensitivity analyses also explored the risk of MD identified by the MDC definition with ≥ 3 points, the risk of 3TC alone during the exposure year (in the absence of either d4T or ddI exposure) and the risk of death for possible MD cases. Odds ratios (OR) and their 95% confidence intervals (CI) were estimated and statistical significance was determined on the basis of Wald Chi Square statistics and an alpha of 0.05. All statistical computations were performed with SAS® 9 .
Of 5703 participants enrolled in protocols 219/219C, 3087 children were perinatally HIV-infected and enrolled by November 3, 2004. Of these children, 156 were ineligible for the present study because they had not been followed for six months or lacked two sequential laboratory evaluations by May 3, 2005, when the dataset was frozen. Thus the final study population included 2931 children. The demographic and clinical characteristics of the study population are shown in Table 1. Fifty-one percent of subjects were female and 91% were 13 years of age or younger at enrollment.
Overall there were 493 incident EPF cases and 556 incident MDC cases with ≥ 2 points. Among the MDC cases, 212 were ≥ 3 point cases; 52 had 4 points. The mean age (standard deviation) for EPF case diagnosis was 8.1 yrs (4.5); MDC ≥ 2 points, 8.8 yrs (4.8), MDC ≥ 3 points, 8.8 (5.0); and MDC 4 points, 9.4 yrs (5.2). For the incident EPF cases, 61.9% presented with CNS conditions and 66.3% with other system conditions; 28.2% had both CNS and other system involvement. For MDC ≥ 2 point cases, the presenting system involvement was muscular for 3.1%, CNS for 42.3%, and multi-system for 54.7%. For ≥3 point cases 3.8% had muscular, 50% CNS, and 46.2% multisystem presentations. For 4 point cases, 57.7% presented with CNS, and 42.3% with multi-system involvement.
Table 2 shows characteristics of participants meeting the incident clinical case definitions used in this study. In utero exposure to antiretroviral medications was documented for 5.9% EPF, 7.1% MDC, and 2.4% EPF/MDC cases. A statistically significant protective association was observed for maternal ARV in utero in EPF cases. MD cases were more likely to have been exposed to postpartum ZDV in the first six weeks of life but this association did not persist in adjusted analysis. Children meeting the EPF, MDC (≥ 2 points and ≥ 3 points), and EPF/MDC case definitions were more likely to have advanced clinical HIV disease at enrollment (CDC clinical category C). CD4% was not associated with MD. VL >100,000/mm3 was associated with MD by the EPF and MDC case definitions, but not for EPF/MDC cases. Finally, there was a reduced risk of MD for each calendar year after 1995.
Table 3 displays the characteristics of study participants according to case definitions of MD. Of 2931 study participants, 768 children (26.2%) had possible MD by the EPF, while 694 (23.7%) met the MDC definition with at least 2 points. Of 694 subjects meeting the MDC, 445 (64.1%) had 2 points, 194 (27.9%) 3 points, and 55 (7.9%) 4 points. Four hundred eighty children (16.4%) met both the EPF and MDC definitions; 58.5% of those were incident cases (Table 3). A higher proportion of MDC cases than EPF cases were incident (80% vs. 64%). Cases were much more likely to die than were non-cases overall (13.7% vs. 2.7%, p < 0.001), with the highest mortality among incident cases meeting both the EPF and MDC (22.1%). When we examined incident cases among all definitions controlling for AUC VL and CD4% as indicators of HIV disease severity, deaths in MD cases were still increased compared to non-cases. Similar to MDC cases overall, MDC cases ≥ 3 points were not associated with CD4% < 15%, but there was increased risk with CDC category C disease and AUC VL > 100,000 copies/ml. Compared to MDC cases overall, a sensitivity analysis showed MDC cases ≥ 3 points were at increased risk of MD with exposure to 3TC (OR 2.55, 95% CI 1.45, 4.50), d4T (OR 2.64, 95% CI 1.50, 4.65), or their combination (OR 1.99 95% CI 1.17, 3.41). Concordance between EPF and MDC for incident possible MD cases was 86.4% (N=2230): 10.9% (N=281) met both criteria and 75.5% (N=1949) met neither case definition.
Figure 1 shows the results of adjusted multiple logistic regression analyses for each NRTI and NRTI combination. There was increased risk of MD by the EPF case definition with 3TC exposure compared to no 3TC exposure (OR 1.53, 95% CI 1.03, 2.27) and with d4T exposure compared to no d4T exposure (OR 2.49, 95% CI 1.64, 3.78) while youths exposed to combination ZDV/ddI were less likely to meet this case definition than those unexposed to ZDV/ddI (OR 0.33, 95% CI 0.17, 0.65). Several ARV exposures increased the risk of MD by the MDC definition compared to youths not exposed to these combinations: 3TC (OR 1.44, 95% CI 1.02, 2.04), d4T (OR 2.34, 95% CI 1.63, 3.36), 3TC/d4T combination (OR 1.58, 95% CI 1.10, 2.26), and d4T/ddI combination (OR 1.73, 95% CI 1.15, 2.60). Two of the targeted medications increased the risk of MD defined by meeting both EPF and MDC definitions: d4T (OR 3.44, 95% CI 1.91, 6.20) and the combination d4T/ddI (OR 2.23, 95% CI 1.19, 4.21). MDC cases ≥ 3 points were at increased risk of MD with exposure to 3TC (OR 2.55, 95% CI 1.45, 4.50), d4T (OR 2.64, 95% CI 1.50, 4.65), or their combination (OR 1.99 95% CI 1.17, 3.41). For all case definitions we found significant associations for exposure to 3TC alone in the year prior to endpoint, as well as exposure to 3TC with either or both d4T and ddI at some point in this exposure year.
In this study we examined the occurrence of clinical and laboratory abnormalities consistent with possible MD in a large prospective cohort of children with perinatally acquired HIV infection according to two published classification schemes. We found that 33.5% of HIV-infected children overall met criteria for possible MD by one of the definitions and 16.4% met criteria by both definitions. This observed frequency of possible MD in HIV-infected children is far greater than has been reported for mitochondrial and respiratory chain disorders in the general population. The minimum birth prevalence of respiratory chain disorders, determined by clinical, enzyme, functional, and molecular criteria, has been estimated to be 13.1/100,000 in Australia . The prevalence of clinically evident mitochondrial DNA disease in adults was 9.2/100,000 adults in northern England, with an additional 16.5/100,000 children and young adults were estimated to be at risk of developing these disorders .
The overall mortality rate among children with possible MD in our study was 13.7%, compared to 2.7% in non-cases and was highest in those children meeting both incident clinical case definitions concurrently (22%). In other studies of children with biochemically and/or molecularly established mitochondrial disorders, but without HIV infection, mortality has been reported to range from 5% to 82% depending on age and clinical manifestations of the children studied. ].
The present study relied on clinical signs since blood lactate/pyruvate measurements or histopathologic, biochemical, genetic or molecular confirmatory testing were not required as part of 219/219C protocol, although the MDC provides a scoring system for such advanced testing when results are available. Thus we have not definitely established that mitochondrial dysfunction was present, nor the mechanism/s responsible for the associations of specific NRTI therapy and possible clinical MD. On the other hand these clinical definitions may prove useful in identifying children who are candidates for specific testing for MD. Here we considered participants meeting the MDC with ≥ 2 points as “cases” for analysis. A recent report describing biopsy results in children screened by the MDC concluded that a clinical score ≥ 3 points was appropriate for pursuing muscle tissue diagnosis for a respiratory chain disorder . 249 MDC cases in this study met the 3 point definition; 212 were incident.
The relatively recent identification of the critical role of mitochondria in cellular processes has likely resulted in under-recognition of the role of mitochondria in a broad range of clinical conditions. Although both NRTI therapy and HIV infection itself are postulated to contribute to MD, the results of this study showed that risk of possible MD was increased in HIV-infected children following NRTI use, after controlling for biologic markers of HIV disease severity. Despite the statistical robustness of the relationship of possible MD to the NRTIs reported here, this study is limited by the lack of histologic, biochemical and molecular analysis of tissue for identifying MD and some children whose abnormalities were not of mitochondrial etiology likely were included. Studies of MD in HIV infection have examined mitochondrial DNA depletion in peripheral blood lymphocytes, however both the assays and the tissues utilized may fail to reflect the mitochondrial status of tissues most affected in HIV-infected children (heart, central nervous system, skeletal muscle, gastrointestinal tract).
A potential limitation of our analytic approach is that only recent ARV exposure (within the previous year) was considered as a determinant of MD, and we did not control for ARV exposures other than that targeted in each analysis. Given that the majority of MD cases who changed regimens during the prior year were still exposed to the target NRTI at year's end, it is likely that associations reported in this study were not substantially affected by misclassification of ARV exposure in relation to the onset of MD. Changes in ARV could have been made in response to signs or symptoms considered to be adverse medication responses or to evidence of disease progression. For children meeting the EPF MD definition, 77% had no ARV regimen change in the prior year, 18% one regimen change, 4% two regimen changes, and 1% had ≥ 3 regimen changes containing any one of the 4 targeted NRTIs. Cases were significantly more likely than non-cases to have changed a NRTI (40% vs. 17% respectively, p 0.001). A similar proportion of cases and non-cases with NRTI changes during the year prior to event had the target exposures at the end of that time period.
More EPF-defined (275) than MDC-defined (138) cases were identified at enrollment, constituting a substantial proportion of all EPF cases (35.8%). The EPF criteria were more inclusive than the 2 point MDC criteria in this cohort, where only 19.9% were deemed to be cases at enrollment, and there were fewer MDC cases overall compared to EPF cases (694 vs. 768). Since about half of the cases in this study met only one definition of possible MD, a substantial number of cases would have been missed had we used a single case definition. The full range of mitochondrial abnormalities in the presence of HIV infection and/or its treatment remains unknown and may not be limited to respiratory chain disorders .
We found possible MD to be associated with recent exposure to NRTIs for all case definitions. These findings are consistent with other studies suggesting that NRTIs, especially d4T, pose particular risks for MD in HIV-infected individuals [14, 42-45]. The finding of increased MD with the NRTIs studied here suggests that pediatric patients should be carefully monitored for signs and symptoms suggesting possible MD when these specific agents or combinations are utilized (d4T, 3TC, 3TC/d4T combination, and d4T/ddI combination).
It has been widely postulated that NRTIs exert their mitochondrial effects through inhibition of DNA polymerase γ , and most studies to date have examined mitochondrial DNA depletion in blood PBMCs or lymphocytes and adipose tissue [21, 47, 48]. Other in vitro and in vivo studies have provided evidence of differential effects of NRTIS and combinations [14-17, 23, 43, 49]. Our study provides support for these studies demonstrating different potentials of individual NRTIs to affect mitochondria [41, 46], and that specific NRTIs in combination may affect mitochondrial function differently than may be predicted by a single NRTI, (e.g. the protective effects of ZDV and ddI in combination in the EPF cases) .
This cohort study is the first to estimate the association between NRTI use and possible MD in a large cohort of children perinatally infected with HIV. As ARV treatment becomes widely available to children in the developing world, varying thresholds for potential mitochondrial toxicities associated with treatment may be observed among children with different genetic backgrounds [10, 11]. The constellation of conditions observed in this study of HIV-infected children receiving NRTIs seriously affects quality of life and even life expectancy. Our results suggest that certain clinical subgroups of children may benefit from specific testing for MD. Further studies to recognize mitochondrial dysfunction in HIV-infected children incorporating histologic, biochemical, and genetic assays are warranted.
We thank the children and families for their participation in PACTG 219C, and the individuals and institutions involved in the conduct of 219C as well as the leadership and participants of the P219/219C protocol team.* We are grateful for the contributions of Joyce Kraimer, Barbara Heckman, Shirley Traite, and Nathan Tryon. Overall support for the International Maternal Pediatric Adolescent AIDS Clinical Trials Group (IMPAACT) was provided by the National Institute of Allergy and Infectious Diseases (U01 AI068632) and the NICHD International and Domestic Pediatric and Maternal HIV Clinical Trials Network supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (contract N01-3-3345 and HHSN267200800001C). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health. This work was supported by the Statistical and Data Analysis Center at Harvard School of Public Health, under the National Institute of Allergy and Infectious Diseases cooperative agreement #5 U01 AI41110 with the Pediatric AIDS Clinical Trials Group (PACTG) and #1 U01 AI068616 with the IMPAACT Group. We also thank the individual staff members and sites who have participated in the conduct of this study, as provided in Appendix I.
Financial support: Grant Number U01AI068632 and 1 U01 AI068616 from the National Institute of Allergy and Infectious Diseases. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases, Eunice Kennedy Shriver National Institute of Child Health and Development, or the National Institutes of Health; Center for Biostatistics in AIDS Research at the Harvard School of Public Health (the statistical and data analysis center of the Pediatric AIDS Clinical Trials Group), under the National Institute of Allergy and Infectious Diseases cooperative agreement No. 5 U01 AI41110; NCRR National Institutes of Health General Clinical Research Center Grant Number RR05096.
Conflict of interest: None of the authors has a commercial or other association that might pose a conflict of interest.
Data presented previously in part at CROI 2008, February 5, 2008, Boston, MA, USA, and published as Abstract #598