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Mitochondrial Dysfunction in HIV+ CNS/Muscular Organ Systems Disorders in HIV+ Children, and perhaps related to accelerated aging in older HIV+ adults
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from Jules: granted the affects in children of mitochondrial dyafunction may be different because their body's are undeveloped when infected while for adults their body has developed already BUT I think this raises the point which I have been trying to make that mitochondrial dysfunction in HIV+ is likely very much related to causing diseases or organ dysfunction in older HIV+ adults who have had HIV & received ARTs for many years including muscular, bone & CNS diseases as well as perhaps this might affect other organ systems including the heart and diabetes. Thus, mitochondrial dysfunction I think plays a crucial role in the accelerated aging process in HIV. I know some researchers don't like the term 'accelerated aging' because they want to have named the precise mechanisms causing the concern which I think is merely mincing words because of course certain disease conditions is causing this. It is very complicated & likely includes senesescence, hepatitis C/B, and activation.
"Studies of mitochondrial dysfunction in HIV infection have examined mitochondrial DNA depletion in peripheral blood lymphocytes, but both the assays and the tissues used may fail to reflect the mitochondrial status of the tissues most affected in HIV-infected children (heart, central nervous system, skeletal muscle, and gastrointestinal tract)......For all case definitions, we found significant associations with exposure to lamivudine alone in the year prior to endpoint, as wll as exposure to lamivudine with either or both stavudine and didanosine at some point in this exposure year......Exposure to several antiretroviral medications increased the risk of mitochondrial dysfunction by the MDC definition compared with children not exposed to these combinations: lamivudine (OR, 1.44 [95% CI, 1.02-2.04]), stavudine (OR, 2.34 [95% CI, 1.63-3.36]), the combination lamivudine-stavudine (OR, 1.58 [95% CI, 1.10-2.26]), and the combination stavudine-didanosine (OR, 1.73 [95% CI, 1.15-2.60])......For the children with incident EPF cases, 305 (61.9%) presented with central nervous system (CNS) conditions and 327 (66.3%) with other system conditions; 139 (28.2%) had both CNS and other system involvement. Among children with MDC cases who had 2 points, the presenting system involvement was muscular for 17 (3.1%), CNS for 235 (42.3%), and multiple-system for 304 (54.7%). Among children with MDC cases who had 3 points, 8 (3.8%) had muscular conditions, 106 (50.0%) had CNS conditions, and 98 (46.2%) had multiple-system presentations. Among children with MDC cases who had 4 points, 30 (57.7%) presented with CNS conditions and 22 (42.3%) presented with multiple-system involvement."
Possible Mitochondrial Dysfunction and Its Association with Antiretroviral Therapy Use in Children Perinatally Infected with HIV
The Journal of Infectious Diseases July 15 2010;202:291-301
Marilyn J. Crain,1 Miriam C. Chernoff,2 James M. Oleske,4 Susan B. Brogly,2 Kathleen M. Malee,5 Peggy R. Borum,6 William A. Meyer III,7 Wendy G. Mitchell,9 John H. Moye,8 Heather M. Ford-Chatterton,10 Russell B. Van Dyke,11 and George R. Seage III2,3
1University of Alabama School of Medicine, Birmingham; 2Center for Biostatistics in AIDS Research and 3Department of Epidemiology, Harvard School of Public Health, Boston, Massachusetts; 4University of Medicine and Dentistry of New Jersey, Newark; 5Children's Memorial Hospital, Chicago, Illinois; 6University of Florida, Gainesville; 7Quest Diagnostics, Baltimore, and 8National Institute of Child Health and Human Development, Bethesda, Maryland; 9University of Southern California School of Medicine, Children's Hospital of Los Angeles, Los Angeles, California; 10Frontier Science and Technology Research Foundation, Amherst, New York; 11Tulane University, New Orleans, Louisiana
ABSTRACT
Background. Mitochondrial dysfunction has been associated with both human immunodeficiency virus (HIV) infection and exposure to antiretroviral therapy. Mitochondrial dysfunction has not been widely studied in HIV-infected children. We estimated the incidence of clinically defined mitochondrial dysfunction among children with perinatal HIV infection.
Methods. Children with perinatal HIV infection enrolled in a prospective cohort study (Pediatric AIDS Clinical Trials Group protocols 219 and 219C) from 1993 through 2004 were included. Two clinical case definitions of mitochondrial dysfunction, the Enquete Perinatale Franaise criteria and the Mitochondrial Disease Classification criteria, were used to classify signs and symptoms that were consistent with possible mitochondrial dysfunction. Adjusted odds ratios of the associations between single and dual nucleoside reverse-transcriptase inhibitor use and possible mitochondrial dysfunction were estimated using logistic regression.
Results. Overall, 982 (33.5%) of 2931 children met 1 or both case definitions of possible mitochondrial dysfunction. Mortality was highest among the 96 children who met both case definitions (20%). After adjusting for confounders, there was a higher risk of possible mitochondrial dysfunction among children who received stavudine regardless of exposure to other medications (odds ratio, 3.44 [95% confidence interval, 1.91-6.20]) or who received stavudine-didanosine combination therapy (odds ratio, 2.23 [95% confidence interval, 1.19-4.21]). Exposure to lamivudine and to lamivudine-stavudine were also associated with an increased risk of mitochondrial dysfunction.
Conclusions. Receipt of nucleoside reverse-transcriptase inhibitors, especially stavudine and lamivudine, was associated with possible mitochondrial dysfunction in children with perinatal HIV infection. Further studies are warranted to elucidate potential mechanisms of nucleoside reverse-transcriptase inhibitor toxicities.
Mitochondrial dysfunction has been reported in children and adults infected with human immunodeficiency virus (HIV). Most studies attribute this finding to antiretroviral medications, specifically nucleoside reverse-transcriptase inhibitors (NRTIs) [1-4]. However, mitochondrial dysfunction has also been reported in HIV-infected individuals who are naive to antiretroviral therapy (ART) [5, 6]. Before an effective prevention or treatment strategy can be devised, it is important to determine the incidence of mitochondrial dysfunction in HIV-infected children and to identify factors associated with its development [6-8].
Studies of the etiology of mitochondrial dysfunction 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 in individuals with confirmed primary mitochondrial disorders [1, 9]. Clinical signs and symptoms of mitochondrial dysfunction may not be recognized in advanced HIV disease, in which marked elevations of HIV viral load are treated with multiple antiretroviral medications. Expression of mitochondrial dysfunction may be related to host mitochondrial haplotypes or other genetic characteristics [10, 11], defects in intergenomic communication such as mitochondrial DNA depletion syndromes [12], environmental influences including HIV infection [13, 14], or specific antiretroviral medications [14-17]. Mitochondrial toxicity due to NRTI use in HIV-infected adults has been reported in patients receiving zidovudine, lamivudine, and stavudine [4, 17-23] and in children who are exposed to didanosine [24, 25]. The main objectives of this study were to estimate the incidence of clinically defined mitochondrial dysfunction among children with perinatal HIV infection and to study its association with exposure to specific antiretroviral medications, specifically NRTIs.
Results
Patient population.Of 5703 participants enrolled in protocols 219 and/or 219C, 3087 children were perinatally infected with HIV and were enrolled by 3 November 2004. Of these children, 156 were ineligible for the present study because they had not been followed up for 6 months or lacked 2 sequential laboratory evaluations by 3 May 2005, when the data set was "frozen." Thus, the final study population included 2931 children. The demographic and clinical characteristics of the study population are shown in Table 1. Of these participants, 1489 (51%) were female and 2675 (91%) were <13 years of age at enrollment.
Overall, there were 493 children with incident EPF cases and 556 children with incident MDC cases who had >2 points. Among the children with MDC cases, 212 had >3 points and 52 had 4 points. The mean age (± standard deviation) for EPF case diagnosis was 8.1 ± 4.5 years, that for MDC case diagnosis with >2 points was 8.8 ± 4.8 years, that for MDC case diagnosis with >3 points was 8.8 ± 5.0 years, and that for MDC case diagnosis with 4 points was 9.4 ± 5.2 years. For the children with incident EPF cases, 305 (61.9%) presented with central nervous system (CNS) conditions and 327 (66.3%) with other system conditions; 139 (28.2%) had both CNS and other system involvement. Among children with MDC cases who had >2 points, the presenting system involvement was muscular for 17 (3.1%), CNS for 235 (42.3%), and multiple-system for 304 (54.7%). Among children with MDC cases who had >3 points, 8 (3.8%) had muscular conditions, 106 (50.0%) had CNS conditions, and 98 (46.2%) had multiple-system presentations. Among children with MDC cases who had 4 points, 30 (57.7%) presented with CNS conditions and 22 (42.3%) presented with multiple-system involvement.
Table 2 shows characteristics of participants whose illness met the incident clinical case definitions used in this study. In utero exposure to antiretroviral medications was documented for 14 (5.9%) of 239 children with EPF cases, 17 (7.1%) of 239 children with MDC cases, and 6 (2.4%) of 249 children with cases defined by both the EPF and MDC criteria. A statistically significant protective association was observed for maternal ART exposure in utero among children with EPF cases. Children with mitochondrial dysfunction were more likely to have had postpartum exposure to zidovudine in the first 6 weeks of life, but this association did not persist in an analysis. Children whose illness met the EPF case definition, those whose illness met the MDC case definition (>2 points), and those whose illness met both the EPF and MDC case definitions were more likely to have advanced clinical HIV disease at enrollment (CDC clinical category C). CD4 cell percentage was not associated with mitochondrial dysfunction. A viral load of >100,000 copies/mL was associated with mitochondrial dysfunction among children whose illness met either the EPF criteria or the MDC criteria, but not among children whose illness met both the EPF and MDC criteria. Finally, there was a reduced risk of mitochondrial dysfunction for each calendar year after 1995.
Incidence of mitochondrial dysfunction.Table 3 displays the proportions of study participants that met the case definitions of mitochondrial dysfunction and the associated mortality rates. Of 2931 study participants, 768 children (26.2%) had possible mitochondrial dysfunction by the EPF definition, whereas 694 (23.7%) met the MDC definition with 2 points. Of 694 children whose illness met the MDC definition, 445 (64.1%) had 2 points, 194 (27.9%) had 3 points, and 55 (7.9%) had 4 points. Four hundred eighty children (16.4%) met both the EPF and MDC definitions; 58.5% of those had incident cases (Table 3). A higher proportion of children with MDC cases than of children with EPF cases had incident cases (80% vs 64%). Case children were much more likely to die than were noncase children overall (13.7% vs 2.7%; p<.001), with the highest mortality among children with incident cases that met both the EPF and MDC definitions (22.1%) (Table 3). When we examined the children with incident cases by all definitions while controlling for area under the curve viral load and CD4 cell percentage as indicators of HIV disease severity, deaths among children with mitochondrial dysfunction were still increased compared with those among noncase children. Similar to MDC cases overall, MDC cases with >3 points were not associated with a CD4 cell percentage of <15%, but there was increased risk with CDC category C disease and an area under the curve viral load of >100,000 copies/mL. Compared with MDC cases overall, a sensitivity analysis showed that MDC cases with >3 points put children at increased risk of mitochondrial dysfunction with exposure to lamivudine (OR, 2.55 [95% CI, 1.45-4.50]), stavudine (OR, 2.64 [95% CI, 1.50-4.65]), or their combination (OR, 1.99 [95% CI, 1.17-3.41]). Concordance between the EPF and MDC criteria for incident possible mitochondrial dysfunction cases was 86.4% (n=2230): 10.9% (n=281) met both sets of criteria and 75.5% (n=1949) met neither case definition.
Association between ART and mitochondrial dysfunction.Figure 1 shows the results of adjusted multiple logistic regression analyses for each NRTI and NRTI combination. There was increased risk of mitochondrial dysfunction by the EPF case definition for lamivudine exposure compared with no lamivudine exposure (OR, 1.53 [95% CI, 1.03-2.27]) and for stavudine exposure compared to no stavudine exposure (OR, 2.49 [95% CI, 1.64-3.78]), whereas children exposed to the combination zidovudine-didanosine were less likely than those unexposed to zidovudine-didanosine to meet this case definition (OR, 0.33 [95% CI, 0.17-0.65]). Exposure to several antiretroviral medications increased the risk of mitochondrial dysfunction by the MDC definition compared with children not exposed to these combinations: lamivudine (OR, 1.44 [95% CI, 1.02-2.04]), stavudine (OR, 2.34 [95% CI, 1.63-3.36]), the combination lamivudine-stavudine (OR, 1.58 [95% CI, 1.10-2.26]), and the combination stavudine-didanosine (OR, 1.73 [95% CI, 1.15-2.60]). Two of the targeted medications increased the risk of mitochondrial dysfunction defined by both the EPF and MDC criteria: stavudine (OR, 3.44 [95% CI, 1.91-6.20]) and the combination stavudine-didanosine (OR, 2.23 [95% CI, [1.19-4.21]). Children with MDC cases who had >3 points were at increased risk of mitochondrial dysfunction with exposure to lamivudine (OR, 2.55 [95% CI, 1.45-4.50]), stavudine (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 with exposure to lamivudine alone in the year prior to endpoint, as well as exposure to lamivudine with either or both stavudine and didanosine at some point in this exposure year.
Discussion
In this study, we examined the occurrence of abnormal clinical and laboratory findings that are consistent with possible mitochondrial dysfunction according to 2 published classification schemes in a large prospective cohort of children with perinatally acquired HIV infection. We found that 33.5% of HIV-infected children overall met the criteria for possible mitochondrial dysfunction by 1 of the definitions and that 16.4% met the criteria by both definitions. This observed incidence of possible mitochondrial dysfunction among HIV-infected children is far greater than the incidence that 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 cases per 100,000 individuals in Australia [35]. The prevalence of clinically evident mitochondrial DNA disease in adults was estimated to be 9.2 cases per 100,000 adults in northern England, with an additional 16.5 cases per 100,000 children and young adults estimated to be at risk of developing these disorders [36].
The overall mortality rate among children with possible mitochondrial dysfunction in our study was 13.7%, compared with a mortality rate of 2.7% among noncase children, and was highest among the children who met 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 be 5%-82% depending on age and clinical manifestations of the children studied [37, 38].
The present study relied on clinical signs because blood lactate and pyruvate measurements or histopathological, biochemical, genetic, or molecular confirmatory testing were not required as part of protocols 219 and 219C, 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 have we established the mechanism or mechanisms responsible for the associations between specific NRTI therapies and possible clinical mitochondrial dysfunction. On the other hand, these clinical definitions may prove useful in identifying children who are candidates for specific testing for mitochondrial dysfunction. Here we considered participants who met the MDC with >2 points as having cases for analysis. A recent report describing biopsy results in children screened by means of the MDC concluded that a clinical score of 3 points was appropriate for pursuing muscle tissue diagnosis for a respiratory chain disorder [39]. In this study, 249 MDC cases 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 underrecognition 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 mitochondrial dysfunction, the results of this study showed that the risk of possible mitochondrial dysfunction was increased among HIV-infected children following NRTI use, after controlling for biological markers of HIV disease severity. Despite the statistical robustness of the relationship between possible mitochondrial dysfunction and the NRTIs reported here, this study is limited by the lack of histological, biochemical, and molecular analysis of tissue for identifying mitochondrial dysfunction, and some children whose abnormalities were not of mitochondrial etiology likely were included. Studies of mitochondrial dysfunction in HIV infection have examined mitochondrial DNA depletion in peripheral blood lymphocytes, but both the assays and the tissues used may fail to reflect the mitochondrial status of the tissues most affected in HIV-infected children (heart, central nervous system, skeletal muscle, and gastrointestinal tract).
A potential limitation of our analytic approach is that only recent ART exposure (within the previous year) was considered to be a determinant of mitochondrial dysfunction, and we did not control for exposure to antiretroviral medications other than that targeted in each analysis. Given that the majority of children with mitochondrial dysfunction who changed regimens during the prior year were still exposed to the target NRTI at the end of the year, it is likely that associations reported in this study were not substantially affected by misclassification of ART exposure in relation to the onset of mitochondrial dysfunction. Changes in ART regime could have been made in response to signs or symptoms that were considered to be adverse medication responses or to evidence of disease progression. For children whose illness met the EPF mitochondrial dysfunction definition, 77% had no ART regimen change in the prior year, 18% had 1 regimen change, 4% had 2 regimen changes, and 1% had >3 regimen changes that contained any 1 of the 4 targeted NRTIs. Case children were statistically significantly more likely than noncase children to have changed a NRTI (40% vs 17%, respectively; p=.001). Similar proportions of case children and noncase children with NRTI changes during the year prior to the mitochondrial dysfunction event were exposed to the target medications at the end of that time period.
More children with EPF-defined cases (275) than children with MDC-defined cases (138) were identified at enrollment, which constituted a substantial proportion of all children with EPF cases (35.8%). The EPF criteria were more inclusive than the 2-point MDC criteria in this cohort, among which only 19.9% were deemed to have cases at enrollment, and there were fewer children with MDC cases overall compared with children with EPF cases (694 vs 768). Because about half of the case children in this study met only 1 definition of possible mitochondrial dysfunction, a substantial number of case children 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 [40].
We found possible mitochondrial dysfunction to be associated with recent exposure to NRTIs for all case definitions. These findings are consistent with those in other studies, which suggests that NRTIs, especially stavudine, pose particular risks for mitochondrial dysfunction in HIV-infected individuals [14, 41-44]. The finding of increased risk of mitochondrial dysfunction for the NRTIs studied here suggests that pediatric patients should be carefully monitored for signs and symptoms that suggest possible mitochondrial dysfunction when these specific agents or combinations are used (stavudine, lamivudine, lamivudine-stavudine combination, and stavudine-didanosine combination).
It has been widely postulated that NRTIs exert their mitochondrial effects through inhibition of DNA polymerase [45], and most studies to date have examined mitochondrial DNA depletion in peripheral blood mononuclear cells or lymphocytes and adipose tissue [21, 46, 47]. Other in vitro and in vivo studies have provided evidence of differential effects of NRTIs and their combinations [14-17, 23, 42, 48]. Our study provides support for these studies that demonstrate different potentials of individual NRTIs to affect mitochondria [40, 45] and that specific NRTIs in combination may affect mitochondrial function differently than may be predicted by a single NRTI (eg, the protective effects of zidovudine and didanosine in combination among the children with EPF cases) [14].
To our knowledge, this cohort study is the first to estimate the association between NRTI use and possible mitochondrial dysfunction in a large cohort of children perinatally infected with HIV. As ART 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 who receive 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 mitochondrial dysfunction. Further studies to recognize mitochondrial dysfunction in HIV-infected children that incorporate histological, biochemical, and genetic assays are warranted.
Methods
Study population.The Pediatric AIDS Clinical Trials Group (PACTG) protocol 219 (Pediatric Late Outcomes study) began on 4 May 1993 as a prospective cohort study to assess the late effects of in utero and neonatal exposure to ART in HIV-exposed children and the effects of ART 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 protocol 219, which ended on 15 September 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 protocol 219 participants with HIV infection enrolled into protocol 219C, in addition to HIV-infected children who were 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 protocol 219 and/or 219C between 4 May 1993 through 3 November 2004 who had at least 2 study visits at 3-month intervals and at least 6 months of laboratory follow-up prior to the closure of the data set on 3 May 2005. Study sites obtained approval from their respective institutional review boards for human research and written consent from the child's parent or guardian.
Data collection. Visits occurred every 3 months until protocol completion, withdrawal, loss to follow-up, or death. At enrollment and during follow-up in protocols 219 and/or 219C, clinical diagnoses and diagnostic test results were recorded and physical examinations and neurological evaluations were performed. Laboratory test results (chemical and hematological analyses), echocardiograms, neuropsychological test results (Bayley Scales of Infant Development and Wechsler tests), and audiometric data were collected according to the protocol schedule or as clinically required. All signs, symptoms, blood chemistry data, and hematological data were assigned a toxicity grade and, together with diagnoses, were coded by professional medical coders with the use of the Medical Dictionary for Regulatory Activities (MedDRA). 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].
Case definitions. Children were considered to have possible mitochondrial dysfunction if they met the criteria for at least 1 of 2 case definitions [32, 33]. The Enquete Perinatale Franaise (EPF) criteria have been used primarily to evaluate possible signs of mitochondrial dysfunction in HIV-uninfected infants who are exposed both to maternal HIV infection and ART in utero but who proved to be free of HIV infection [28, 32]. Children who met this definition had at least 1 major condition (neurological or involving another organ system) at any visit or 2 minor conditions reported on at least 2 visits [32].
The Mitochondrial Disease Classification (MDC) criteria were developed as a clinical tool that uses 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 [33]. Points are assigned for 40 diagnoses according to the primary clinical presentation: muscular (maximum of 2 points), central nervous system (maximum of 2 points), or multiple systems (maximum of 3 points), with additional points added for subsequent clinical conditions, to a maximum of 4 points for clinical findings. For >4 points to be accumulated, additional specified testing for mitochondrial disorders, including metabolic, laboratory, and tissue abnormalities, is required [33]. Children with 2 points may then be evaluated further with invasive tests, including muscle biopsy for histological and metabolic testing, for further point accumulation to definitively establish a respiratory chain defect. Because protocols 219 and 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 the category of possible mitochondrial dysfunction within the MDC criteria) were considered to have cases of possible mitochondrial dysfunction. Both clinical and laboratory criteria for possible mitochondrial dysfunction required the development of definitions for persistence (>3 months) 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 abnormal clinical and laboratory findings who met the criteria for the EPF and MDC case definitions separately were identified. Diagnoses or laboratory values that triggered case definition conditions were systematically reviewed. The primary function of the review was to eliminate diagnoses, terms, and laboratory values for which an alternative explanation to mitochondrial dysfunction was plausible (eg, neurosensory hearing loss in a child with confirmed cytomegalovirus infection). Clinicians were blinded to ART 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 on which the participant reached his or her maximum MDC score was considered to be the date of mitochondrial dysfunction diagnosis. For the EPF criteria, the date of mitochondrial dysfunction diagnosis was the date of the earliest event that met the case criteria. Children with prevalent cases of mitochondrial dysfunction were those whose event date was on or prior to the date of study enrollment.
ART use.ART use was classified according to individual NRTI and specific NRTI combinations. Single NRTI agents commonly used as a component of ART were considered (lamivudine, zidovudine, stavudine, and didanosine), as well as common NRTI combinations (lamivudine-stavudine, zidovudine-didanosine, zidovudine-lamivudine, and stavudine-didanosine). ART use in the year prior to the presentation of clinical signs of possible mitochondrial dysfunction or in the year prior to the end of study follow-up if no mitochondrial dysfunction event occurred was included.
Statistical analysis. The prevalence and incidence of mitochondrial dysfunction by the EPF and MDC definitions were estimated. For concordance of incident events, children with cases that were either positive or negative for both the EPF and MDC criteria were evaluated. Children with cases that met either case definition prior to study entry were considered to have prevalent cases and were excluded from the incidence analysis.
Logistic regression was used to estimate the association between ART use and incident mitochondrial dysfunction, while controlling for potential confounders that were identified a priori: sex, race and ethnicity, age at study entry, neonatal zidovudine prophylaxis, maternal ART exposure (prenatal and intrapartum), Centers for Disease Control and Prevention (CDC) clinical category at study entry [34], time-adjusted area under the curve HIV RNA load, and most recent CD4 cell percentage prior to the year of ART use. CDC clinical category, viral load, and CD4 cell percentage were included because HIV disease severity may relate to both ART use and possible mitochondrial dysfunction. The standard of care for HIV-infected children evolved during the study period; thus, the logistical analysis also included the number of years before (negative) or after (positive) 1995 prior to the mitochondrial dysfunction event or the end of follow-up.
The association between mitochondrial dysfunction and ART was estimated for each of 4 single NRTIs and 4 NRTI combinations. In each analysis, the referent group comprised children who were not receiving the antiretroviral medication being studied during the year prior to event or censor time, although they may have received other antiretroviral medications during this exposure year. Sensitivity analyses also explored the risk of mitochondrial dysfunction (identified by 3 points in the MDC definition), the risk of mitochondrial dysfunction for lamivudine alone during the exposure year (in the absence of either stavudine or didanosine exposure), and the risk of death for possible mitochondrial dysfunction cases. Odds ratios (ORs) and their 95% confidence intervals (CIs) were estimated, and statistical significance was determined on the basis of Wald χ 2 statistics and an value of 0.05. All statistical computations were performed with SAS software (version 9.1; SAS Institute).
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