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 [36
]. 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 [37
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. [38
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 [40
]. 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 [41
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
]. 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 [46
], and most studies to date have examined mitochondrial DNA depletion in blood PBMCs or lymphocytes and adipose tissue [21
]. Other in vitro
and in vivo
studies have provided evidence of differential effects of NRTIS and combinations [14
]. Our study provides support for these studies demonstrating different potentials of individual NRTIs to affect mitochondria [41
], 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) [14
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
]. 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.