antiviral therapy9:849–863 review complications associated

The availability of durable, effective antiretroviral therapyfor HIV-infected patients has fundamentally altered theprognosis of this disease and has also increased aware-ness that long-term drug toxicities have the potential tocause significant morbidity and even mortality in thispatient population. The long-term use of nucleosideanalogue reverse transcriptase inhibitor (NRTI) drugs hasbeen associated with a number of clinically relevant toxi-cities including hyperlactataemia and lactic acidosis,

neuropathy, pancreatitis and, more recently, a syndromeof pathological loss of subcutaneous fat tissue (lipoat-rophy). Importantly, the toxicity profile of each NRTI drugwithin this class is unique in terms of the overall risk oflong-term complications, as well as the tissue specificityof its toxic effects. In this review, the clinical manifesta-tions, risk factors and pathological basis forNRTI-associated toxicity syndromes are explored, with anemphasis on clinical assessment and management.

Review

Complications associated with NRTI therapy: update on clinical features and possible pathogenicmechanisms David Nolan and Simon Mallal*

Centre for Clinical Immunology and Biomedical Statistics, Royal Perth Hospital and Murdoch University, Western Australia, Australia

*Corresponding author: Tel: +61 89 244 2899; Fax: +61 89 244 2920; E-mail: [email protected]

Antiviral Therapy 9:849–863

Among the clinically available antiretroviral drugs,nucleoside analogue reverse transcriptase inhibitors(NRTIs) were the first to demonstrate clinical efficacyagainst HIV infection. NRTIs continue to be used incontemporary highly active antiretroviral therapy(HAART) regimens where they may be given in combina-tion with HIV protease inhibitors (PIs) or non-nucleosidereverse transcriptase inhibitors (NNRTIs). The ongoinguse of NRTI therapy into the second decade of HIVtherapy highlights the need for a comprehensive under-standing of NRTI-induced drug toxicities [1–3], as HIVinfection becomes a manageable disease with greatlyreduced morbidity and mortality attributable to immunedeficiency [4]. Given current estimates that approxi-mately 40 million individuals are HIV-infectedworldwide, including more than 1.5 million individualsin the United States and Western Europe who are likelyto receive long-term antiretroviral therapy [5] (seealso http://www.unaids.org/worldaidsday/2002/press/Epiupdate), understanding and managing these adverseeffects appropriately are of significant importance.

Mode of action of NRTI drugs

The therapeutic activity of NRTI drugs is conferred bytheir ability to inhibit HIV reverse transcriptase,

the viral RNA-dependent DNA polymerase that allowsthe virus to create a DNA sequence based on its ownRNA template (Figure 1). Following intracellular phos-phorylation, NRTI drugs compete with naturallyoccurring nucleotides for utilization by HIV reversetranscriptase. Incorporation of the ‘false’ (NRTI)substrate then causes DNA chain termination, as thesenucleotides lack a hydroxyl group that is critical forchain elongation. The effectiveness of NRTI drugs asinhibitors of HIV replication is therefore determinedprimarily by their ability to inhibit HIV reverse tran-scriptase. However, the efficiency with which NRTIdrugs are delivered to the appropriate cell populationand intracellular compartment – determined by factorssuch as oral bioavailability, cellular transport mecha-nisms and the efficiency of cellular kinases responsiblefor activating NRTI drugs to their triphosphate form –is also an important factor in determining drug efficacy.

Clinicians will be familiar with the concept thatNRTI drugs act directly to reduce HIV-RNA levels intreated patients and that successful therapy is associ-ated with markedly reduced viral load early in thetreatment course (that is, within 1–6 months [6]).Subsequent improvements in CD4+ T-cell counts andimmunological function occur more gradually, asreduction in the viral burden allows immune function

Introduction

©2004 International Medical Press 1359-6535/02/$17.00 849

to be restored as a secondary phenomenon. As will beargued in this review, an approach to management thatrecognizes the distinction between immediate effects oftreatment (viral load reduction) and later effects onother disease outcomes (for example, CD4+ T-cellcounts and progression to AIDS) is one that may alsobe applied to NRTI-associated toxicity.

Potential mechanisms of NRTI-mediatedmitochondrial toxicity

The study of NRTI-associated toxicity syndromes, nowmore than a decade old, has also been informed by thepharmacology of these drugs [1–3], particularly bytheir capacity to inhibit host mitochondrial DNA(mtDNA) polymerase-gamma through a mechanismthat is similar to their therapeutic activity as HIVreverse transcriptase inhibitors. However, it is worthstating at the outset that the toxic and therapeuticprofiles of individual NRTI drugs are not correlated, sothat an increased efficacy is not necessarily accompa-nied by increased toxicity [7]. Indeed, over the past 15years there has been an increasing trend towards NRTI

drugs that combine high levels of antiviral efficacy withbenign toxicity profiles both in vitro and (thus far) invivo, providing cause for optimism that the prevalenceof NRTI-associated toxicities will decline over time.This trend towards reduced toxicity may also reflectthe avoidance of NRTI combinations that do havesynergistic toxicity profiles, as may be seen in the caseof NRTI-associated neuropathy where stavudine anddidanosine each contribute to increased risk. This willbe discussed further below.

Mitochondria are the energy powerhouses of the celland are unique among intracellular organelles incontaining their own genome that is extrachromo-somal – replicating independently of nuclear DNA.Hence, mtDNA can be synthesized in post-mitotic andresting cells, utilizing a unique DNA polymerase(polymerase-gamma) whose evolutionary origins [8]and enzymatic activities [9] are distinct from themultiple DNA polymerases involved in nuclear DNAsynthesis. Importantly, DNA polymerase-gamma isalso unique among the human polymerases in that it isunable to discriminate between naturally occurringnucleotides and nucleoside analogue drugs [8]. Hence,

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©2004 International Medical Press850

Figure 1. The basis of NRTI-associated mitochondrial toxicity according to the ‘pol-gamma’ model

NRTI drugs differ from natural nucleoside compounds in that the 5′-hydroxyl group (indicated by arrow) is modified or removed, so that incorporation into a nascentDNA chain leads to chain termination. Note that the affinity of a given NRTI drug for DNA polymerase-gamma (a determinant of its ability to cause mtDNA deple-tion) is not related to its affinity for HIV reverse transcriptase (a determinant of antiretroviral efficacy). NRTI-MP, NRTI-monophosphate; NRTI-DP, NRTI-diphosphate;NRTI-TP, NRTI-triphosphate.

O

NRTI-MP NRTI-DP NRTI-TP

HN

O

(OH)

CH3

≠Toxicity

DNA polymerase-gamma HIV reverse transcriptase

dsDNA ssDNA RNA

Efficacy

NRTI

NRTIs are capable of inhibiting cellular mtDNAsynthesis via their effects on DNA polymerase-gamma(Figure 1). The proposal that NRTI-induced poly-merase-gamma inhibition represents a commonpathway for tissue-specific adverse effects of this drugclass has been enunciated by Lewis & Dalakas [10]and by Brinkman et al. [11], and reviewed morerecently by Kakuda [2] and White [3]. The basicpremise of the ‘pol-gamma’ hypothesis is that NRTI-induced inhibition of this DNA polymerase leads tocellular mtDNA depletion (that is, reduced numbers ofmtDNA copies per cell). Cellular, and ultimatelyorgan, toxicity is the consequence of loss of mito-chondrial bioenergetic function, when mtDNA levelshave fallen beyond a critical level at which the produc-tion of the 13 mtDNA-encoded protein subunits of the

mitochondrial respiratory chain is insufficient to meetenergy requirements (see Figure 2).

At the cellular level, there is an increasing under-standing that while the ability of a specific NRTI toinduce mtDNA depletion is primarily determined by itsrelative affinity for polymerase-gamma, the relevanceof this effect to the development of tissue toxicity ismodulated by other factors, such as that drug’s abilityto access the mitochondrial compartment within thecell cytosol in an activated form. This is highlighted bythe recent discovery of a mitochondrial innermembrane transporter that is able to facilitate the entryof active NRTI diphosphate and triphosphate deriva-tives into mitochondria [12].

Is the inhibition of mtDNA polymerase-gamma asufficient explanation for all NRTI-associated toxicity

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Complications of NRTI therapy

Figure 2. The mitochondrial organelle and its role in oxidative phosphorylation

Mitochondria are ubiquitous organelles responsible for cellular energy production through oxidative phosphorylation. This requires the concerted action of mtDNA-encoded subunits (13) and nuclear DNA-encoded subunits (~70) of the five respiratory chain complexes. Note also that all proteins involved in determiningmitochondrial organellar number and structure, as well as in maintaining biochemical pathways within the mitochondrial matrix (for example, beta-oxidation, Krebs’cycle), are nuclear DNA-encoded.

Mitochondrion(5–10 mitochondrialgenomes/organelle)

Cell(100s of mitochondria)

Complex I II III IV VTotal protein subunits 43 4 10 13 12MtDNA-encoded subunits 7 0 1 3 2

Mitochondrialrespiratory chain activity

Acetyl-CoA

NADH FADH2

TCAcycle

H+ H+

H+H+

H+H+

H+

Electrontransportchain

ADPATP

ADPATP

glucose + fatty acids

β-oxidation

Oxidative metabolism

syndromes? The probable answer to this question isno, although alternative explanations appear to be theexception rather than the rule. For example, zidovu-dine may exert at least some of its toxic effects through‘non-pol-gamma’ mechanisms, with a body of researchevidence indicating that zidovudine has direct effectson critical components of the mitochondrial membraneinvolved in cellular energy metabolism (for example,the ADP/ATP transporter that exports mitochondrial-derived ATP to the cytosol) [13–15]. This is animportant consideration when designing studiesseeking to identify mitochondrial effects of selectNRTIs, as mitochondrial dysfunction (reflected byincreased lactate production, decreased activity ofmitochondrial respiratory complexes, or both) mayoccur in the absence of, or before, mtDNA depletion.

It is also important to recognize that inhibitingmtDNA polymerase-gamma may affect the fidelity ofmtDNA synthesis – resulting in increased risk ofmtDNA mutation – as well as causing mtDNA deple-tion [16,17]. The clinical importance of this effect hasnot been established, although it appears to be a rela-tively rare cause of disease. Nevertheless, neurologicalsyndromes attributable to mtDNA mutations havebeen described in association with NRTI therapy,including five published cases of Leber’s hereditaryoptic neuropathy (characterized by rapidly evolvingbilateral optic atrophy and blindness) [18–21]. It islikely that these individuals were genetically suscep-tible to this condition, in which the development of theclinical phenotype generally requires a very highproportion of mutated mtDNA, and that NRTItherapy may have ‘unmasked’ the syndrome. However,the development of an unusual neurological syndromein an NRTI-treated individual should alert the clinicianto the possibility of a ‘mitochondrial disease’. Similarly,mtDNA mutation may be considered in rare cases inwhich mitochondrial dysfunction appears to beprogressive despite cessation of NRTI therapy [22].

Mitochondrial toxicity and pregnancy: foetaland maternal considerations

Before returning to more typical manifestations ofmitochondrial toxicity associated with NRTI therapy,there has been concern that foetal exposure to NRTItherapy in utero may lead to the development of mito-chondrial disease in infants, following the report ofeight children with consistent clinical features [23]. Acomprehensive US epidemiological study examiningthis issue [n=1954, including 188 deaths at age <5years, with evidence of mitochondrial dysfunctionrated from ‘unlikely’ (Class 1) to ‘highly likely/proven’(Class 4)] has not identified increased mortality attrib-utable to mitochondrial disease among NRTI-exposed

infants [24], and concluded that the benefits of peri-natal zidovudine therapy are likely to outweigh therisks [24,25]. However, the French investigators whoinitially described this association have gone on todemonstrate an 18-month incidence of ‘established’mitochondrial dysfunction (that is, clinical syndromesupported by histopathology and evidence of mito-chondrial respiratory chain defects) among ~0.3%infants in a similarly large population-based study(7/2644 cases among NRTI-exposed infants, 0/1748 inunexposed controls) [26]. ‘Possible’ mitochondrialdysfunction was identified in a further 14 NRTI-exposed infants. This study also identified a relativelycoherent clinical syndrome, consisting of i) neurolog-ical symptoms (developmental delay, seizures,behavioural disturbances), ii) cerebral magnetic reso-nance imaging abnormalities (white matter andbrainstem lesions) and iii) hyperlactataemia. Riskfactor analysis including both ‘possible’ and ‘estab-lished’ cases suggested increased risk associated withcombined NRTI therapy (15/21 cases; zidovudine +lamivudine, n=14) compared with zidovudinemonotherapy (6/21 cases) (RR=2.5, 95% CI 1.0–6.5).Intravenous zidovudine therapy was utilized in 20/21cases. However, duration of NRTI therapy was notfound to be different among cases and controls andmtDNA depletion was not found in affected tissuesdespite the presence of histopathological and biochem-ical evidence of mitochondrial toxicity. The authorsstressed that these findings do not argue against theusefulness of NRTI therapy in pregnancy [26].

With respect to the risks of maternal mitochondrialtoxicity associated with NRTI use in pregnancy,however, there is increasing evidence that stavudinetherapy – particularly when combined with didanosine– is associated with risk of lactic acidosis in late preg-nancy [27,28]. This prompted the manufacturers toissue product information in 2001 warning that thecombination of stavudine and didanosine should beavoided during pregnancy ‘unless the potential benefitclearly outweighs the potential risks’ [29].

In vitro studies: establishing toxicity profilesfor NRTI drugs

In vitro studies have provided important informationregarding the potential of specific NRTI drugs to causeDNA polymerase-gamma inhibition, mtDNA deple-tion and mitochondrial dysfunction [9,30–32]. Aconsistent ranking of the potency of mtDNA synthesisinhibition has emerged from these studies, withdecreasing potency associated with zalcitabine (ddC)>> didanosine (ddI) > stavudine (d4T) > zidovudine(AZT). These studies also suggest that no significantmitochondrial polymerase-gamma inhibition occurs at

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pharmacological concentrations of lamivudine,abacavir and tenofovir. One caveat to these findings isthat NRTI-associated mitochondrial toxicity is tissue-specific, so that in vitro studies should ideally utilizehuman cell cultures that are relevant to the clinicaltoxicity profile of the drug.

These studies can also shed light on the intracellularactivation pathways of specific drugs, which may alsoinfluence their ability to affect mitochondria (Figure 3).For example, the thymidine analogues zidovudine andstavudine share a common activation pathway thatinvolves thymidine kinase (TK) in the formation of themonophosphate form of the drug. TK comprises twoisoforms: TK1, which is cytosolic and active in mitoti-cally active cells only and TK2, which ismitochondria-specific and active in post-mitotic cells.Of interest, stavudine – but not zidovudine – retainsanti-HIV activity within the pharmacological doserange in TK1-deficient cells (plasma IC50 for HIV=0.27

µM with TK and 2.5 µM in TK-deficient CEM cells)[33], and is efficiently phosphorylated to its activetriphosphate derivative within isolated mitochondria[34]. This suggests that this thymidine analogue ismore likely to target mtDNA, particularly in post-mitotic tissues (for example, nerves), in which mtDNAsynthesis accounts for a greater proportion of nucleo-side utilization while nuclear DNA synthesis is latent.

Clinical NRTI-induced toxicity syndromes

While the prototypic NRTI-associated toxicitysyndrome is zidovudine myopathy, this syndrome isnow rare in clinical practice [35,36], possibly reflectingeither the use of lower zidovudine doses in the HAARTera or the involvement of uncontrolled HIV infectionper se in the pathogenesis of this syndrome. Currently,the adverse effects of most concern involving NRTItherapy are lactic acidosis and other less serious



Figure 3. NRTI activation pathways and mitochondrial partitioning

The intracellular metabolism of two commonly used thymidine analogue NRTI drugs zidovudine and stavudine. The relative ability of these drugs to gain access tothe mitochondrial compartment and subsequently inhibit mtDNA synthesis is determined by the efficiency and rate of kinase-mediated phosphorylation to the activetriphosphate form, as well as their relative affinity for DNA polymerase-gamma. Note also that the conversion of free drug to the monophosphate derivative is irre-versible, whilst subsequent steps are reversible. TK1, thymidine kinase 1; TK2, thymidine kinase 2.

O

TK1

TK1

TK2

TK2

ZDV-DPZDV-TP

ZDV-TPZDV-DPZDV-MP

(~7%)

d4T-MP

d4T-MP

d4T-DP

d4T-DP d4T-TP

dNT carrier

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CH3

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NH3

d4T-TP(~35%)

~45%

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disorders of lactate metabolism, the progressive subcu-taneous fat wasting syndrome that is viewed as part ofthe lipodystrophy syndrome, neuropathy and pancre-atitis (Figure 4).

Lactic acidosis and hyperlactataemia Lactic acidosis is probably the most recognizablefeature of mitochondrial dysfunction in clinical disease,in which loss of mitochondrial oxidative function leadsto increased reliance on anaerobic metabolism and theinevitable accumulation of lactate (and thus, of acid). Inthe setting of NRTI therapy, there is now an apprecia-tion of a spectrum of clinical phenotypes associatedwith elevated systemic lactate levels (Figure 5) [37]. Atone end of this spectrum is a relatively commonsyndrome of mild, asymptomatic, non-progressivehyperlactataemia (generally <2.5 mmol/l), whichappears to represent a ‘compensated’ homeostaticsystem in which elevated lactate production is balancedby effective mechanisms of lactate clearance. While thedegree of hyperlactataemia appears to be greater in thepresence of stavudine or didanosine therapy comparedwith zidovudine or abacavir, this syndrome appears tobe benign, irrespective of the choice of NRTI therapy[38–42].

In contrast, lactic acidosis and hepatic steatosisrepresents a relatively uncommon (approx 1–2 per1000 person-years) but life-threatening clinicalsyndrome in which lactate homeostasis is completely‘decompensated’, allowing the rapid, progressive accu-mulation of lactate and development of acidosis inaffected patients. A critical aspect of lactic acidosis is itsunpredictability, as it typically occurs in patients whohave been on stable NRTI regimens for months or evenyears, and is not heralded by increased lactate levelsbefore the development of the fulminant syndrome[38,43]. Host risk factors have been identified forNRTI-associated lactic acidosis, including concurrentliver disease, female gender and obesity. While themajority of reported cases in recent years have involvedstavudine-based HAART, it is important to recognizethat cases involving zidovudine therapy also occur(reviewed in [37,43]). The cornerstone of managementof this condition is early recognition of the clinicalmanifestations: abdominal symptoms (includingnausea, vomiting, anorexia, abdominal pain and disten-sion) and fatigue, with biochemical evidence ofhepatocellular liver dysfunction and lactate levelgenerally >5 mmol/l. These clinical and laboratoryabnormalities should lead to prompt cessation of NRTI

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Figure 4. Clinical syndromes attributed to NRTI-associated mitochondrial toxicity

Tissue-specificmitochondrial toxicity

syndromes

Myopathy: Associated with AZTMyalgia, weaknessElevated creatine kinaseVery rare in clinical practice

Pancreatitis: Associated with d4T and/or ddICentral abdominal painElevated amylase/lipase~1 case/100 patient-years

Lipoatrophy: Associated with d4T > AZTFat loss: especially legs, face > trunkMeasurable with DEXA* scans– subclinical changes common No biochemical featuresCommon: d4T ~40%, AZT~15%at 30 months

Neuropathy: Associated with ddC > ddI,d4TPainful sensory neuropathyAbnormal electrophysiology– subclinical changes commonNo biochemical features Common: ddC/ddI/d4T 40%

Non-tissue-specificmitochondrial toxicity

syndromes

Leber neuropathy describedBilateral optic neuritisLikely in geneticallysusceptible individuals, mayhave positive family history

Adult ‘mitochondrial disease' syndromes:

Lactic acidosis: Associated with NRTIs Abdominal pain, lethargy, tachypnea, neuromuscular weakness May be asymptomatic Often unheralded, acute onset Elevated lactate, ↓HCO3 ~0.1 case/100 patient-years High mortality: ~30%

Developmental delay, seizuresMRI abnormalitiesHyperlactataemiaRare: <0.3% 18-month incidencePrognosis uncertain

Infantile NRTI toxicity syndrome:

*DEXA, dual-energy X-ray absorptiometry scans.

therapy. A more recently identified clinical syndromeaccompanying lactic acidosis is progressive, severeneuromuscular weakness mimicking the Guillain-Barrésyndrome [44]. Initial reports by the US Food and DrugAdministration identified 25 cases, including seven fatalcases (notably, NRTI therapy was continued after thediagnosis was made in all but one of these individuals).A more recent retrospective analysis has identified 55cases (44 associated with stavudine therapy), with a25% mortality rate among the 36 patients with docu-mented follow-up [45]. Overall, the mortalityassociated with NRTI-associated lactic acidosis remainsunacceptably high at around 50% [43].

Given the association between lactic acidosis andhepatic steatosis, it is worth noting that hepaticsteatosis is not an infrequent finding in liver biopsysamples obtained from HIV-infected patients whodevelop abnormal liver function tests on long-termantiretroviral therapy, where it may represent acommon element in a number of disparate disorders(for example, alcoholic and non-alcoholic steatohep-atitis, diabetes mellitus). Hence, this finding is neitherhighly predictive of lactic acidosis nor is it specific tothis syndrome.

An ‘intermediate’ syndrome has also been described,characterized by symptomatic hyperlactataemia orhepatic steatosis without systemic acidosis. This

syndrome is almost uniformly associated with stavu-dine therapy, with an incidence of approximately 13per 1000 person-years [46,47]. Of importance, there isevidence that lactate levels and symptoms can becontrolled following modification of NRTI therapy tozidovudine or abacavir [48].

With regards to the pathogenesis of hyperlac-tataemia syndromes, evidence has been provided fromstudies using exogenous lactate loading that increasedlactate production is the fundamental defect in chronicasymptomatic hyperlactataemia, rather than decreasedclearance of lactate, which primarily involves the liver[49]. Indeed, lactate clearance appears to be up-regulated as a compensatory response to an elevatedsystemic lactate load [49]. The source of this excessivelactate production is unknown, although a carefullyconducted exercise physiology study indicates thatskeletal muscle is an unlikely candidate [50]. Incontrast, lactic acidosis represents a complete failure oflactate homeostasis, in which lactic acid accumulationprogresses inexorably while NRTI therapy is main-tained. In this scenario, the prominent role of livermitochondrial dysfunction (indicated by the presenceof microvesicular hepatic steatosis) [37,43,46] leadingto a shift from hepatic lactate consumption to lactateproduction would be predicted to have dramatic conse-quences for systemic lactate metabolism.



Figure 5. The spectrum of disease associated with NRTI-associated hyperlactataemia

Lactate in mmol/l

1 2 3 4 5 6

'Compensated' hyperlactataemia versus

chronicstable over timeHCO ≥20 mmol/lcommonrisk factors – NRTIs, d4T>ZDV

rapidly progressivelife-threateningHCO3 <20 mmol/lrarerisks – women, HCV/HBV co-infection, liver disease

'Decompensated' lactic acidosis/hepatic steatosis

Asymptomatichyperlactataemia

Symptomatichyperlactataemia

Lacticacidosis

Mortality30–50%

Côté and colleagues have examined mtDNA deple-tion in peripheral blood mononuclear cells in eightpatients with symptomatic hyperlactataemia, using areal-time polymerase chain reaction (PCR) quantitationmethod [51]. All patients were receiving stavudinetherapy at the time of their hyperlactataemia syndrome(stavudine/didanosine, n=6; stavudine/lamivudine, n=2).This study demonstrated significant mtDNA depletionin blood in all affected patients compared withuntreated HIV-infected controls, which resolved aftercessation of NRTI therapy (all cases) and remainednormal after resumption of alternative NRTI regimensin four cases (abacavir/lamivudine, n=2; zidovudine/lamivudine, n=1; lamivudine only, n=1). This group hasalso found mtDNA depletion of similar magnitude inpatients without symptomatic lactataemia who wereenrolled in the SWATCH study and were receivingstavudine/didanosine therapy, while zidovudine/lamivu-dine therapy reduced mtDNA content to a lesser degree[52]. Thus, it remains to be determined whetheranalysis of blood mtDNA depletion provides indepen-dent information about the risk of symptomatichyperlactataemia in stavudine-treated patients, orwhether the choice of NRTI therapy is the major deter-minant of mtDNA depletion in blood, irrespective ofthe clinical phenotype.

LipoatrophyThere is now a weight of evidence that the choice andduration of NRTI drugs represents the dominant riskfactor for developing pathological loss of fat tissue, aclinical outcome referred to as subcutaneous fatwasting or lipoatrophy. The importance of treatmentchoices in determining the risk of developing lipoat-rophy is highlighted by the fact that this long-termcomplication is extremely rare in the general populationas well as in treatment-naive HIV-infected individuals.Hence, there are no known modifiable risk factors fordeveloping lipoatrophy that are present in the generalpopulation. This may be contrasted with other aspectsof the broader clinical syndrome commonly referred toas lipodystrophy, which also includes metabolic compli-cations such as dyslipidaemia and insulin resistance,and the visceral/abdominal fat accumulation that oftenaccompanies these metabolic changes. These metaboliccomplications of therapy, which have a much strongerassociation with HIV PI therapy than with NRTIdrugs, are also highly prevalent in the general popula-tion, suggesting that risk may be modified by‘non-drug’ factors such as diet and physical activity.This topic has been reviewed elsewhere [79] and willnot be addressed here.

Clinical studies have demonstrated that NRTItherapy alone provides sufficient conditions for thedevelopment of lipoatrophy [53–55] and that NRTI

therapy is an independent risk factor for its occurrencein HAART-treated individuals [56–58]. Clinical trialsdata have now confirmed the findings of observationalcohort studies (reviewed in [59]) that stavudinetherapy is associated with an approximately twofoldincreased risk of lipoatrophy compared with zidovu-dine, so that the risk of clinically apparent lipoatrophyover 30 months is approximately 10–20% in zidovu-dine-treated individuals and 40–50% among thosetreated with stavudine [60–62]. Significant differencesin limb fat between stavudine and zidovudine treat-ment arms in the ACTG 384 trial have also beendocumented from 48 weeks onwards [63]. Finally,switching NRTI therapy from stavudine to abacavir inan observational study [64] and more recently in tworandomized trials [65,66], has been associated withsignificant improvements in fat wasting. In contrast,over 30 trials investigating HIV PI discontinuation as atherapeutic strategy (reviewed in [67]) have failed todemonstrate beneficial effects on fat wasting, althoughmetabolic abnormalities such as dyslipidaemia andinsulin resistance did improve. It is notable, however,that improvement in fat wasting at 48 weeks appearsto be modest at best, with absolute increases in limb fatof approximately 1% [65]. Therefore, it would seemprudent to focus on identifying HAART regimens witha low risk of inducing fat wasting, since restoration ofestablished fat wasting appears to be a slow andpossibly incomplete process.

The histopathological correlates of this syndromealso indicate that adipose tissue-specific mitochondrialtoxicity is central to lipoatrophy pathogenesis. At theultrastructural level, abnormal mitochondrial architec-ture and organellar proliferation are prominentfeatures [68–70], while increased adipocyte apoptosisassociated with lipoatrophy has been shown toimprove after discontinuing stavudine therapy infavour of abacavir [71], but not after switching HIV PItherapy [72]. Mitochondrial DNA depletion in adiposetissue samples has also been documented using semi-quantitative techniques [70,73] and in studies ofadipose tissue using precise real-time PCR quantitationmethods, which have demonstrated significant mtDNAdepletion particularly associated with stavudinecompared with zidovudine [74,75]. Further evidencethat mitochondrial dysfunction is a direct consequenceof NRTI therapy was provided by Patrick Mallon ofthe University of New South Wales, Sydney, Australia,in an oral presentation at the 11th Conference onRetroviruses & Opportunistic Infections [115]. In thisimportant, albeit small study, it was found that 2 weeks’therapy with stavudine/lamivudine (n=10) was suffi-cient to cause significantly reduced expression of severalmitochondrial gene products [for example, cytochromeoxygenase (COX)1, COX3 and cytochrome b mRNA]

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in HIV-negative volunteers (all P<0.02). Effects ofzidovudine/lamivudine treatment (n=10) were lessmarked, with significant reduction of COX3 mRNAonly (P=0.04).

Finally, our own group has attempted to link thecausal events between NRTI therapy, adipocytemtDNA depletion, adipose tissue toxicity and the even-tual development of clinical fat loss. In a publishedstudy [76], we have been able to demonstrate thatstavudine therapy was associated with more severeadipocyte mtDNA depletion (P<0.001) and fat wastingover time (P=0.002) compared with zidovudinetherapy in independent analyses. Among a subset ofpatients with concurrent biopsy and longitudinal bodycomposition data (dual-energy X-ray absorptiometryscans), fat wasting was associated with duration ofNRTI therapy (P=0.001) and adipocyte mtDNAcopies/cell (P=0.01). In this analysis, the associationbetween choice of stavudine versus zidovudine and fatwasting (P=0.03) was no longer significant afteradjustment for the effect of mtDNA depletion(P=0.13), providing evidence that the ability of thesedrugs to cause fat loss manifests specifically throughmtDNA depletion. Confocal microscopy analysis alsosupported this conclusion, demonstrating consistentrelationships between severity of adipose tissue toxicityand mtDNA depletion. No significant effects of HIV PItherapy were detected in these analyses. In a subse-quent study, we have also shown that adipocytemtDNA depletion is associated with loss of mitochon-drial function [77].

The role of NRTI drugs other than stavudine andzidovudine as risk factors for lipoatrophy has beenmore difficult to discern, particularly as lamivudineand didanosine are generally used in combination withthese thymidine analogue drugs. Thus far, no indepen-dent effect of these ‘second’ NRTI drugs has emerged,with results from the available clinical trials completedto 30 months showing similar risk of lipoatrophy ifstavudine is combined with didanosine or with lamivu-dine [60–62]. Data regarding the concurrent use ofzidovudine and didanosine is more difficult to obtain,although an early study of dual NRTI therapy by Saint-Marc and colleagues [53] included a number of patientson didanosine in stavudine (d4T/ddI=13/27, 48%) andzidovudine treatment groups (AZT/ddI=13/16, 81%)that were well-matched for NRTI therapy duration[53]. In this study, use of stavudine remained the mostsignificant risk factor for lipoatrophy (relative risk 1.95compared with zidovudine), while no significantdidanosine effect could be demonstrated. Use ofdidanosine compared with lamivudine also had nosignificant effect on adipose tissue mtDNA depletion ina recent analysis of 232 biopsy samples after adjust-ment for the effect of stavudine treatment [75]. In

terms of the newer drugs abacavir (an NRTI) and teno-fovir (a nucleotide RTI), which would be predicted tohave reduced predisposition to cause mitochondrialtoxicity [30,31], clinical trials data are limited to lessthan 2 years of follow-up at this stage, but show noevidence for an association with lipoatrophy. Indeed,96-week data from the Gilead 903 study involvingcomparisons of tenofovir and stavudine therapy(combined with lamivudine and efavirenz in botharms), objectively measured limb fat was reduced byapproximately 40% in the stavudine arm comparedwith those on tenofovir [78].

NeuropathyThere are a number of issues to be considered inaddressing the possible associations between NRTItherapy, mitochondrial toxicity and neuropathy.Firstly, neuropathic changes frequently do not come tothe attention of clinicians as the symptoms are ofgradual onset and clinical signs of nerve damage arenot generally sought in clinical practice. Hence, theprevalence – and potential long-term impact – ofneuropathy is likely to be underestimated. Secondly,there is a definite contribution of HIV disease per se tothe pathogenesis of a form of distal sensory neuropathythat is clinically and electrophysiologically indistin-guishable from so-called ‘toxic neuropathy fromantiretroviral drugs’ (reviewed in [79]). It is thereforedifficult to dissect out the relative contribution ofdisease-associated and drug-associated factors in thesyndrome, as these effects are likely to be synergistic.The clinical syndrome common to both HIV-associatedand toxic sensory polyneuropathy is dominated byperipheral pain and dysaesthaesia, with rare motorinvolvement.

In general, the relative risk of toxic polyneuropathyassociated with specific NRTI drugs correlates wellwith their ability to inhibit mtDNA polymerase-gammain vitro. The greatest risk has been associated withzalcitabine, an NRTI with equivalent affinity formtDNA polymerase-gamma and HIV reverse transcrip-tase, thus providing for a narrow therapeutic window[9]. Neuropathy was a consistent and dose-limitingtoxicity in trials of zalcitabine, occurring in approxi-mately one-third of patients receiving low-dose therapy(2.25 mg/day) for less than a year [80,81], while higherdoses were associated with almost universal develop-ment of neuropathy (100% with >0.04 mg/kg/day and80% with 0.04 mg/kg/day) [80]. Consistent with the‘pol-gamma’ hypothesis, Dalakas and colleagues [82]have provided in vivo evidence of significant mtDNAdepletion in peripheral nerves associated with thiscomplication of zalcitabine therapy.

Stavudine and didanosine have also been associatedwith increased risk of neuropathy in the large Johns



Hopkins AIDS Service cohort study (n=1116).Compared with a crude incidence rate of 6.8 cases per100 person-years for didanosine, stavudine use wasassociated with a relative risk of 1.4, while concurrentuse of these drugs further increased risk (3.5-fold rela-tive risk for stavudine/didanosine compared withdidanosine alone). Concurrent hydroxyurea therapyhad an additional effect (7.8-fold relative risk of stavu-dine/didanosine/hydroxyurea compared with didano-sine alone, giving a crude incidence of 28.6 cases per100 person-years) [83].

As already mentioned, markers of progressive HIVdisease also correlate with the development of sensorypolyneuropathy, with evidence for associationsbetween reduced CD4 T-cell counts and increased riskof neuropathy (reviewed in [84]), and increased painscores in patients with higher HIV viral load [85].However, in a cohort of 144 ambulatory patients withlonger duration of exposure to stavudine and/ordidanosine and average CD4 T-cell counts >400×106/l,the prevalence of sensory neuropathy was found to be44% [86], suggesting that among patients withprolonged survival and effective immunological andvirological outcomes, choice and duration of NRTItherapy will ultimately become the dominant riskfactor for neuropathy. In this analysis, use of a ‘d’ drug(that is, stavudine, didanosine or zalcitabine) was asso-ciated with an odds ratio of 26 of developing clinicalneuropathy, while disease-associated factors were notfound to be significant in multivariate analysis.

Consistent with the lipoatrophy data, reversal ofestablished neuropathy appears to be a slow process thatis dependent on cessation of the offending NRTI drug.The identification of L-carnitine deficiency and the use ofL-acetyl-carnitine therapy may also have beneficialeffects on nerve regeneration [82,83]. Lamotriginetherapy has also been associated with improved painsymptoms in the context of NRTI-associated toxicpolyneuropathy [79]. As with any clinical neuropathy, asearch for contributing factors such as diabetes,excessive alcohol consumption and vitamin deficiencies(for example, thiamine, B12 and folate) should be under-taken [89]. Ideally, these factors should also beconsidered prior to initiating NRTI therapy.

PancreatitisThere is a differential diagnosis for pancreatitis occurr-ing in an HIV-infected individual that includesNRTI-associated disease, in which case pancreatitismay be an isolated event or may occur in the contextof systemic lactic acidosis [90]. Other causes must alsobe considered including pancreatitis secondary tosevere HIV PI-induced hypertriglyceridaemia [91,92]and the effects of other drugs such as pentamidine [93].In this review, pancreatitis will be considered as a

diagnosis that includes clinical symptoms of abdominalpain combined with elevated serum amylase levels.This excludes cases of isolated hyperamylasaemia,which is increased in prevalence among HIV-infectedpatients with moderate to severe immune deficiency[94], as the relationship between this biochemicalabnormality and pancreatitis is uncertain.

The most comprehensive data concerning riskfactors for NRTI-associated pancreatitis again comefrom the Johns Hopkins AIDS Service cohort (n=2613,33 cases) [90]. In this analysis, didanosine and stavu-dine were associated with roughly equivalent risk ofpancreatitis, with estimated incidence rates of 0.8and 1.1 cases per 100 person-years, respectively.Zidovudine therapy was associated with an incidenceof 0.1 per 100 person-years. Combining stavudine anddidanosine increased risk approximately twofold,while the concurrent use of didanosine and hydroxy-urea increased relative risk approximately eightfold[90]. Risk associated with didanosine and stavudinetherapy was also highlighted in the ACTG 5025 study,which involved switching to a regimen ofstavudine/didanosine/indinavir with or withouthydroxyurea (n=68 in each arm) compared withongoing zidovudine/lamivudine/indinavir (n=66) instable, aviraemic patients with CD4 T-cell countsgreater than 200×106/l. Stavudine and didanosinetreatment was associated with a 4% incidence ofpancreatitis (three in each arm), including three fatalcases in patients receiving hydroxyurea [95].

Preventing NRTI-associated mitochondrialtoxicities

From the data already presented, it is apparent thatNRTI drugs are associated with differential risks ofmitochondrial toxicity and that these toxicitysyndromes are tissue-specific. The selection of NRTIdrugs to minimize risk of toxicity is therefore ofprimary importance. However, in the clinical settingthis is obviously not a straightforward decision whenthere are other factors at play such as drug resistanceand the potential risk of other adverse effects such asabacavir hypersensitivity. In this regard, there has beenprogress towards identifying a highly predictive geneticmarker of the abacavir hypersensitivity syndromewithin the human leukocyte antigen region [96].

In the case of tenofovir, the potential for renal toxi-city remains a concern in the long-term, given that thistissue-specific toxicity was observed with anothernucleotide analogue (adefovir [97]), and that both ofthese drugs can be transported across the renal tubularmembrane by a specific protein (human renal organicanion transporter 1) [98]. The manufacturers of teno-fovir have carefully examined the effects of this drug in

Nolan & Mallal


human proximal tubular cells, as well as other tissues invitro [31], finding no evidence of cellular or mitochon-drial toxicity across a range of doses (3–300 µM).However, clinical monitoring is still warranted, as twopublished case reports have identified acute renal failurein tenofovir recipients – one with background chronicstable renal impairment [99], while the other casedisplayed hallmark features of renal tubular toxicity(Fanconi syndrome, nephrogenic diabetes insipidus andacute renal failure) [100]. Whether these rare eventsrepresent mitochondrial toxicity is unknown.

A common feature of these mitochondrial toxicitysyndromes, with the exception of lactic acidosis, is thatrisk is increased in patients with advanced immunedeficiency at the commencement of antiretroviraltherapy (that is, with CD4 T-cell counts <200×106/l).This is true for lipoatrophy [101] as it is forneuropathy [84] and pancreatitis [90]. Hence, delayingthe introduction of antiretroviral therapy does notseem supportable as a strategy for preventing NRTI-associated toxicity.

Otherwise, the recognition of other factors that maycontribute independently to risk of toxicity syndromes isalso important. This is most relevant to neuropathy andpancreatitis, where the risk factors in the general popu-lation are well known. In the case of lipoatrophy, nomodifiable host risk factors have emerged, although riskof this complication is increased in those of white raceand older age [102]. No adjuvant therapies haveemerged that may prevent or decrease the toxic effects ofNRTI drugs. On the other hand, there have been reportsthat risk of mitochondrial toxicity is increased whenNRTI therapy is combined with ribavirin and inter-feron-alpha for the treatment of hepatitis C co-infection[103–105]. This is certainly plausible, as ribavirin is apurine analogue [106] (like didanosine), while inter-feron-alpha may inhibit mtDNA transcription [107].

Monitoring NRTI-associated mitochondrialtoxicities

As a general principle, the identification and manage-ment of mitochondrial toxicities associated with NRTItherapy depend on clinical assessment rather than theuse of laboratory markers. It is therefore importantthat clinicians are aware of the contribution of NRTItherapy to specific toxicity syndromes, as well as therelevant clinical manifestations of these syndromes.Perhaps even more importantly, the early recognitionof toxicities such as lipoatrophy and neuropathy (byboth clinician and patient) is critical if there is to be arealistic expectation that these adverse effects canbe reversed.

Several research groups have developed highly preciseassays for the measurement of mtDNA depletion in

tissue samples and blood [51,106,107]. These assaysare an attractive diagnostic tool, as the mtDNA levelper cell is likely to reflect the direct consequences ofNRTI therapy in affected tissue early in the treatmentcourse, which may therefore predict the subsequentprogression of tissue pathology to clinically apparentdisease over time. This may be seen as analagous to therole of HIV viral load in determining the rate of diseaseprogression. However, there is not a widespread clin-ical application for these tools at present. Afundamental aspect of these syndromes is their tissuespecificity, so that while important data can beobtained from the relevant target tissue (that is,adipose tissue in lipodystrophy studies and peripheralnerve tissue or neurites in neuropathy studies), theseeffects are not reflected in more readily obtainedsamples such as blood samples. For example, in arecent study involving 62 patients who had concurrentblood and adipose tissue samples collected at 6-monthly intervals (160 study visits in total) [75],mtDNA depletion was strongly associated with stavu-dine use, but not didanosine, in adipose tissue(P=0.001 and P=0.71, respectively). In contrast,didanosine therapy was the dominant determinant ofperipheral blood mtDNA depletion (P=0.07), whilestavudine therapy had no significant effect (P=0.80).The specific association between peripheral bloodmtDNA depletion and the use of didansoine and/orzalcitabine (but not zidovudine or stavudine) has alsobeen documented in other studies [111–113].

Similarly, routine measurement of systemic lactatelevels does not appear to allow prediction of the moresevere forms of lactic acidosis or of tissue-specificNRTI toxicities [114], although routine screening oflactate levels in pregnant women receiving NRTItherapy does seem warranted. Hence, even in hyper-lactataemia syndromes (in which biochemical ratherthan clinical assessment would appear logical), theimportance of clinical syndrome recognition cannotbe overstated.

It is also important to recognize that the clinicalmanifestations of NRTI-associated toxicity, whichform the basis of diagnostic classification systems, arelikely to represent advanced and potentially irre-versible tissue pathology. In this regard, activeassessment for symptoms and signs according to thespecific toxicity profile of a given NRTI drug is essen-tial in order to identify reversible disease.

Conclusions and future prospects

The benefits of highly active antiretroviral regimens areclear. Although novel approaches to HIV therapy arealways on the horizon, we are now entering a ‘secondHAART era’ in which new compounds are being



developed within current drug classes (includingNRTIs) that retain their antiviral properties whilstminimizing toxicity. It is anticipated that the lessonsthat have been learned regarding the pathogenesis oftoxicity syndromes – particularly those associated withNRTI therapy, in which the ‘pol-gamma’ hypothesishas been generally vindicated – will be applied to drugdesign in the future. Certainly, the limited and slowreversal of many tissue-specific toxicities followingremoval of the offending agent (for example, fatwasting and neuropathy) suggests that it is far moreprudent to prevent these syndromes through the use ofrelatively non-toxic drugs rather than to attempt totreat and reverse them once they are established.

References1. Squires KE. An introduction to nucleoside and nucleotide

analogues. Antiviral Therapy 2001; 6(Suppl. 3):1–14.

2. Kakuda TN. Pharmacology of nucleoside and nucleotidereverse transcriptase inhibitor-induced mitochondrial toxi-city. Clinical Therapy 2000; 22:685–708.

3. White AJ. Mitochondrial toxicity and HIV therapy.Sexually Transmitted Infections 2001; 77:158–173.

4. Palella FJ Jr, Delaney KM, Moorman AC, Loveless MO,Fuhrer J, Satten GA, Aschman DJ & Holmberg SD. TheHIV Outpatient Study Investigators. Declining morbidityand mortality among patients with advanced humanimmunodeficiency virus infection. New England Journal ofMedicine 1998; 338:853–860.

5. Piot P, Bartos M, Ghys PD, Walker N & Schwartlander B.The global impact of HIV/AIDS. Nature 2001; 410:968–973.

6. Chene G, Sterne JA, May M, Costagliola D, Ledergerber B,Phillips AN, Dabis F, Lundgren J, D’Arminio Monforte A,de Wolf F, Hogg R, Reiss P, Justice A, Leport C, StaszewskiS, Gill J, Fatkenheuer G & Egger ME; for theAntiretroviral Therapy Cohort Collaboration. Prognosticimportance of initial response in HIV-1 infected patientsstarting potent antiretroviral therapy: analysis of prospec-tive studies. Lancet 2003; 362:679–686.

7. Martin JL, Brown CE, Matthews-Davis N & Reardon JE.Effects of antiviral nucleoside analogs on human DNApolymerases and mitochondrial DNA synthesis.Antimicrobial Agents & Chemotherapy 1994;38:2743–2749.

8. Burgers PM, Koonin EV, Bruford E, Blanco L, Burtis KC,Christman MF, Copeland WC, Friedberg EC, Hanaoka F,Hinkle DC, Lawrence CW, Nakanishi M, Ohmori H,Prakash L, Prakash S, Reynaud CA, Sugino A, Todo T,Wang Z, Weill JC & Woodgate R. Eukaryotic DNA poly-merases: proposal for a revised nomenclature. Journal ofBiological Chemistry 2001; 276:43487–43490.

9. Longley MJ, Ropp PA, Lim SE & Copeland WC.Characterization of the native and recombinant catalyticsubunit of human DNA polymerase-gamma Identificationof residues critical for exonuclease activity and dideoxynu-cleotide sensitivity. Biochemistry 1998; 37:10529–10539.

10. Lewis W & Dalakas MC. Mitochondrial toxicity ofantiviral drugs. Nature Medicine 1995; 1:417–422.

11. Brinkman K, ter Hofstede HJ, Burger DM, Smeitink JA &Koopmans PP. Adverse effects of reverse transcriptaseinhibitors: mitochondrial toxicity as common pathway.AIDS 1998; 12:1735–1744.

12. Dolce V, Fiermonte G, Runswick MJ, Palmieri F & WalkerJE. The human mitochondrial deoxynucleotide carrier andits role in the toxicity of nucleoside antivirals. Proceedingsof the National Academy of Sciences, USA 2001;98:2284–2288.

13. Hobbs GA, Keilbaugh SA, Rief PM & Simpson MV.Cellular targets of 3′-azido-3′-deoxythymidine: an early(non-delayed) effect on oxidative phosphorylation.Biochemical Pharmacology 1995; 50:381–390.

14. Barile M, Valenti D, Quagliarello E & Passarella S.Mitochondria as cell targets of AZT (zidovudine). GeneralPharmacology 1998; 31:531–538.

15. Masini A, Scotti C, Calligaro A, Cazzalini O, Stivala LA,Bianchi L, Giovannini F, Ceccarelli D, Muscatello U,Tomasi A & Vannini V. Zidovudine-induced experimentalmyopathy: dual mechanism of mitochondrial damage.Journal of Neurological Sciences 1999; 166:131–140.

16. Lewis W, Copeland WC & Day BJ. Mitochondrial DNAdepletion, oxidative stress, and mutation: mechanisms ofdysfunction from nucleoside reverse transcriptaseinhibitors. Laboratory Investigation 2001; 81:777–790.

17. Martin AM, Hammond E, Nolan D, Pace C, Den Boer M,Taylor L, Moore H, Martinez OP, Christiansen FT &Mallal S. Accumulation of mitochondrial DNA mutationsin human immunodeficiency virus-infected patients treatedwith nucleoside-analogue reverse-transcriptase inhibitors.American Journal of Human Genetics 2003; 72:549–560.[Erratum published in American Journal of HumanGenetics 2003 72:1358.]

18. Luzhansky JZ, Pierce AB, Hoy JF & Hall AJ. Leber’shereditary optic neuropathy in the setting of nucleosideanalogue toxicity. AIDS 2001; 15:1588–1589.

19. Shaikh S, Ta C, Basham AA & Mansour S. Leber heredi-tary optic neuropathy associated with antiretroviraltherapy for human immunodeficiency virus infection.American Journal of Ophthalmology 2001; 131:143–145.

20. Warner JE & Ries KM. Optic neuropathy in a patient withAIDS. Journal of Neuroophthalmology 2001; 21:92–94.

21. Mackey DA, Fingert JH, Luzhansky JZ, McCluskey PJ,Howell N, Hall AJ, Pierce AB & Hoy JF. Leber’s hereditaryoptic neuropathy triggered by antiretroviral therapy forhuman immunodeficiency virus. Eye 2003; 17:312–317.

22. Carr A, Morey A, Mallon P, Williams D & Thorburn DR.Fatal portal hypertension, liver failure, and mitochondrialdysfunction after HIV-1 nucleoside analogue-inducedhepatitis and lactic acidaemia. Lancet 2001;357:1412–1414.

23. Blanche S, Tardieu M, Rustin P, Slama A, Barret B, FirtionG, Ciraru-Vigneron N, Lacroix C, Rouzioux C,Mandelbrot L, Desguerre I, Rotig A, Mayaux MJ &Delfraissy JF. Persistent mitochondrial dysfunction andperinatal exposure to antiretroviral nucleoside analogues.Lancet 1999; 354:1084–1089.

24. Bulterys M, Nesheim S, Abrams EJ, Palumbo P, Farley J,Lampe M & Fowler MG for the Perinatal Safety ReviewWorking Group. Lack of evidence of mitochondrialdysfunction in the offspring of HIV-infected women.Retrospective review of perinatal exposure to antiretroviraldrugs in the Perinatal AIDS Collaborative TransmissionStudy. Annals of the New York Academy of Sciences 2000;918:212–221.

25. Barret B, Tardieu M, Rustin P, Lacroix C, Chabrol B,Desguerre I, Dollfus C, Mayaux MJ & Blanche S for theFrench Perinatal Cohort Study Group. Persistent mitochon-drial dysfunction in HIV-1-exposed but uninfected infants:clinical screening in a large prospective cohort. AIDS 2003;17:1769–1785.

26. Tuomala RE, Shapiro DE, Mofenson LM, Bryson Y,Culnane M, Hughes MD, O’Sullivan MJ, Scott G, StekAM, Wara D & Bulterys M. Antiretroviral therapy duringpregnancy and the risk of an adverse outcome. NewEngland Journal of Medicine 2002; 346:1863–1870.

27. Mandelbrot L, Kermarrec N, Marcollet A, Lafanechere A,Longuet P, Chosidow D & Saada M. Case report: nucleo-side analogue-induced lactic acidosis in the third trimesterof pregnancy. AIDS 2003; 17:272–273.

28. Sarner L & Fakoya A. Case report: acute onset lacticacidosis and pancreatitis in the third trimester of pregnancyin HIV-1 positive women taking antiretroviral medication.Sexually Transmitted Infections 2002; 78:58–59.

Nolan & Mallal




29. Bristol-Myers Squibb Company. Healthcare Provider –Important Drug Warning Letter. 5 January 2001. NewYork: Bristol-Myers Squibb.

30. Johnson AA, Ray AS, Hanes J, Suo Z, Colacino JM,Anderson KS & Johnson KA. Toxicity of antiviral nucleo-side analogs and the human mitochondrial DNApolymerase. Journal of Biological Chemistry 2001;276:40847–40857.

31. Birkus G, Hitchcock MJ & Cihlar T. Assessment of mito-chondrial toxicity in human cells treated with tenofovir:comparison with other nucleoside reverse transcriptaseinhibitors. Antimicrobial Agents & Chemotherapy 2002;46:716–723.

32. Walker UA, Setzer B & Venhoff N. Increased long-termmitochondrial toxicity in combinations of nucleosideanalogue reverse-transcriptase inhibitors. AIDS 2002;16:2165–2173.

33. Turriziani O, Simeoni E, Dianzani F & Antonelli G. Anti-HIV antiviral activity of stavudine in a thymidinekinase-deficient cellular line. Antiviral Therapy 1998;3:191–194.

34. Cui L, Locatelli L, Xie M-Y & Sommadossi JP. Effect ofnucleoside analogs on neurite regeneration and mitochon-drial DNA synthesis in PC-12 cells. Journal ofPharmacology & Experimental Therapeutics 1997;280:1228–1234.

35. Manfredi R, Motta R, Patrono D, Calza L, Chiodo F &Boni P. A prospective case-control survey of laboratorymarkers of skeletal muscle damage during HIV disease andantiretroviral therapy. AIDS 2002; 16:1969–1971.

36. Simpson DM, Katzenstein DA, Hughes MD, Hammer SM,Williamson DL, Jiang Q & Pi JT. Neuromuscular functionin HIV infection: analysis of a placebo-controlled combina-tion antiretroviral trial. AIDS 1998; 12:2425–2432.

37. John M & Mallal S. Hyperlactatemia syndromes in peoplewith HIV infection. Current Opinion in Infectious Diseases2002; 15:23–29.

38. John M, Moore CB, James IR, Nolan D, Upton RP,McKinnon EJ & Mallal SA. Chronic hyperlactatemia inHIV-infected patients taking antiretroviral therapy. AIDS2001; 15:717–723.

39. Boubaker K, Flepp M, Sudre P, Furrer H, Haensel A,Hirschel B, Boggian K, Chave JP, Bernasconi E, Egger M,Opravil M, Rickenbach M, Francioli P & Telenti A.Hyperlactatemia and antiretroviral therapy: the Swiss HIVCohort Study. Clinical Infectious Diseases 2001;33:1931–1937.

40. Moyle GJ, Datta D, Mandalia S, Morlese J, Asboe D &Gazzard BG. Hyperlactataemia and lactic acidosis duringantiretroviral therapy: relevance, reproducibility andpossible risk factors. AIDS 2002; 16:1341–1349.

41. Vrouenraets SM, Treskes M, Regez RM, Troost N,Smulders YM, Weigel HM, Frissen PH & Brinkman K.Hyperlactataemia in HIV-infected patients: the role ofNRTI-treatment. Antiviral Therapy 2002; 7:239–244.

42. Hocqueloux L, Alberti C, Feugeas JP, Lafaurie M,Lukasiewicz E, Bagnard G, Carel O, Erlich D & MolinaJM. Prevalence, risk factors and outcome of hyperlac-tataemia in HIV-infected patients. HIV Medicine 2003;4:18–23.

43. Falco V, Rodriguez D, Ribera E, Martinez E, Miro JM,Domingo P, Diazaraque R, Arribas JR, Gonzalez-Garcia JJ,Montero F, Sanchez L & Pahissa A. Severe nucleoside-asso-ciated lactic acidosis in human immunodeficiencyvirus-infected patients: report of 12 cases and review of theliterature. Clinical Infectious Diseases 2002; 34:838–846.

44. Wooltorton E. HIV drug stavudine (Zerit, d4T) and symp-toms mimicking Guillain–Barre syndrome. CanadianMedical Association Journal 2002; 166:1067.

45. Simpson D, Estanislao L, Marcus K, Truffa M, McArthurJ, Lucey B, Dorfman D, Naismith R, Guerrero I, Mendez J,Lonergan T, Landau Y, Harris M, Montaner J & CliffordD. HIV-associated neuromuscular weakness syndrome.10th Conference on Retroviruses & Opportunistic

Infections. 10–14 February 2003. Boston, Mass., USA.Abstract 87.

46. Gerard Y, Maulin L, Yazdanpanah Y, De La Tribonniere X,Amiel C, Maurage CA, Robin S, Sablonniere B, DhennainC & Mouton Y. Symptomatic hyperlactataemia: anemerging complication of antiretroviral therapy. AIDS2000; 14:2723–2730.

47. Lonergan JT, Behling C, Pfander H, Hassanein TI &Mathews WC. Hyperlactatemia and hepatic abnormalitiesin 10 HIV-infected patients receiving nucleoside analoguecombination regimens. Clinical Infectious Diseases 2000;31:162–166.

48. Lonergan T, McComsey G, Fisher R, Shalit P, File T, WardD, Williams V, Lindsey L & Hernandez J. Improvements insymptomatic hyperlactatemia are observed after 12 weekswhen stavudine is replaced by either abacavir or zidovu-dine. Antiviral Therapy 2001; 6(Suppl. 4):55–56.

49. Leclercq P, Roth H, Bosseray A & Leverve X. Investigatinglactate metabolism to estimate mitochondrial status.Antiviral Therapy 2001; 6(Suppl. 4):16.

50. Roge BT, Calbet JA, Moller K, Ullum H, Hendel HW,Gerstoft J & Pedersen BK. Skeletal muscle mitochondrialfunction and exercise capacity in HIV-infected patientswith lipodystrophy and elevated p-lactate levels. AIDS2002; 16:973–982.

51. Côté HCF, Brumme ZL, Craib KJP, Alexander CS,Wynhoven B, Ting L, Wong H, Harris M, Harrigan PR,O’Shaughnessy MV & Montaner JSG. Changes in mito-chondrial DNA as a marker of nucleoside toxicity inHIV-infected patients. New England Journal of Medicine2002; 346:811–820.

52. Cote HC, Yip B, Asselin JJ, Chan JW, Hogg RS, HarriganPR, O’Shaughnessy MV & Montaner JS.Mitochondrial:nuclear DNA ratios in peripheral blood cellsfrom human immunodeficiency virus (HIV)-infectedpatients who received selected HIV antiretroviral drug regi-mens. Journal of Infectious Diseases 2003;187:1972–1976.

53. Saint-Marc T, Partisani M, Poizot-Martin I, Bruno F,Rouviere O, Lang JM, Gastaut JA & Touraine JL. Asyndrome of peripheral fat wasting (lipodystrophy) inpatients receiving long-term nucleoside analogue therapy.AIDS 1999; 13:1659–1667.

54. Madge S, Kinloch-de-Loes S, Mercey D, Johnson MA &Weller IV. Lipodystrophy in patients naive to HIV proteaseinhibitors. AIDS 1999; 13:735–737.

55. Carr A, Miller J, Law M & Cooper DA. A syndrome oflipoatrophy, lactic acidemia and liver dysfunction associ-ated with HIV nucleoside analogue therapy: contributionto protease inhibitor-related lipodystrophy syndrome.AIDS 2000; 14:F25–F32.

56. Mallal SA, John M, Moore CB, James IR & McKinnon EJ.Contribution of nucleoside analogue reverse transcriptaseinhibitors to subcutaneous fat wasting in patients with HIVinfection. AIDS 2000; 14:1309–1316.

57. Saint-Marc T, Partisani M, Poizot-Martin I, Rouviere O,Bruno F, Avellaneda R, Lang JM, Gastaut JA & TouraineJL. Fat distribution evaluated by computed tomographyand metabolic abnormalities in patients undergoing anti-retroviral therapy: preliminary results of the LIPOCOstudy. AIDS 2000; 14:37–49.

58. van der Valk M, Gisolf EH, Reiss P, Wit FW, Japour A,Weverling GJ & Danner SA for the Prometheus StudyGroup. Increased risk of lipodystrophy when nucleosideanalogue reverse transcriptase inhibitors are included withprotease inhibitors in the treatment of HIV-1 infection.AIDS 2001; 15:847–855.

59. John M, Nolan D & Mallal S. Antiretroviral therapy andthe lipodystrophy syndrome. Antiviral Therapy 2001;6:9–20.

60. Chene G, Angelini E, Cotte L, Lang JM, Morlat P,Rancinan C, May T, Journot V, Raffi F, Jarrousse B,Grappin M, Lepeu G & Molina JM. Role of long-termnucleoside-analogue therapy in lipodystrophy and metabolic

disorders in human immunodeficiency virus-infectedpatients. Clinical Infectious Diseases 2002; 34:649–657.

61. Amin J, Moore A, Carr A, French MA, Law M, Emery S& Cooper DA; OzCombo 1 and 2 Investigators. Combinedanalysis of two-year follow-up from two open-labelrandomized trials comparing efficacy of three nucleosidereverse transcriptase inhibitor backbones for previouslyuntreated HIV-1 infection: Ozcombo 1 and 2. HIV ClinicalTrials 2003; 4:252–261.

62. Joly V, Flandre P, Meiffredy V, Leturque N, Harel M,Aboulker JP & Yeni P. Increased risk of lipoatrophy understavudine in HIV-1-infected patients: results of a substudyfrom a comparative trial. AIDS 2002; 16:2447–2454.

63. Dubé MP, Zackin R, Tebas P, Roubenoff R, Mulligan K,Robbins G, Yang Y, Shafer R, Snyder S & Grinspoon SK.Prospective study of regional body composition in anti-retroviral-naive subjects randomized to receivezidovudine+lamivudine or didanosine+stavudine combinedwith nelfinavir, efavirenz, or both: A5005s, a substudy ofACTG 384. Antiviral Therapy 2002; 7:L18.

64. Saint-Marc T & Touraine JL. The effects of discontinuingstavudine therapy on clinical and metabolic abnormalitiesin patients suffering from lipodystrophy. AIDS 1999;13:2188–2189.

65. Carr A, Workman C, Smith DE, Hoy J, Hudson J, DoongN, Martin A, Amin J, Freund J, Law M & Cooper DA forthe Mitochondrial Toxicity (MITOX) Study Group.Abacavir substitution for nucleoside analogs in patientswith HIV lipoatrophy: a randomized trial. Journal of theAmerican Medical Association 2002; 288:207–215.

66. Lafeuillade A, Clumeck N, Mallolas J, Jaeger H, LivrozetJM, Ferreira Mdo S, Johnson M, Cheret A & Antoun Zfor the European Trizal team. Comparison of metabolicabnormalities and clinical lipodystrophy 48 weeks afterswitching from HAART to Trizivir versus continuedHAART: the Trizal study. HIV Clinical Trials 2003;4:37–43.

67. Dreschler H & Powderly WG. Switching effective anti-retroviral therapy: a review. Clinical Infectious Diseases2002; 35:1219–1230.

68. Nolan D, Hammond E, Martin A, Taylor L, Herrmann S,McKinnon E, Metcalf C, Latham B & Mallal S.Mitochondrial DNA depletion and morphologic changes inadipocytes associated with nucleoside reverse transcriptaseinhibitor therapy. AIDS 2003; 17:1329–1338.

69. Lloreta J, Domingo P, Pujol RM, Arroyo JA, Baixeras N,Matias-Guiu X, Gilaberte M, Sambeat MA & Serrano S.Ultrastructural features of highly active antiretroviraltherapy-associated partial lipodystrophy. Virchows Archiv2002; 441:599–604.

70. Walker UA, Bickel M, Lutke Volksbeck SI, Ketelsen UP,Schofer H, Setzer B, Venhoff N, Rickerts V & StaszewskiS. Evidence of nucleoside analogue reverse transcriptaseinhibitor-associated genetic and structural defects of mito-chondria in adipose tissue of HIV-infected patients. Journalof Acquired Immune Deficiency Syndromes 2002;29:117–121.

71. Thompson K, McComsey G, Paulsen D, Cherry C,Lonergan T, Hessenthaler S, Williams V, Fisher R,Wesselingh S, Hernandez J & Ross L. Improvements inbody fat and mitochondrial DNA levels are accompaniedby decreased adipose tissue apoptosis after replacement ofstavudine therapy with either abacavir or stavudine. 10thConference on Retroviruses & Opportunistic Infections.10–14 February 2003, Boston, Mass., USA. Abstract 728.

72. Domingo P, Matias-Guiu X, Pujol RM, Domingo JC,Arroyo JA, Sambeat MA & Vazquez G. Switching to nevi-rapine decreases insulin levels but does not improvesubcutaneous adipocyte apoptosis in patients with highlyactive antiretroviral therapy-associated lipodystrophy.Journal of Infectious Diseases 2001; 184:1197–1201.

73. Shikuma CM, Hu N, Milne C, Yost F, Waslien C, ShimizuS & Shiramizu B. Mitochondrial DNA decrease in subcuta-neous adipose tissue of HIV-infected individuals withperipheral lipoatrophy. AIDS 2001; 15:1801–1809.

74. Nolan D, Hammond E, James I, McKinnon E & Mallal S.Contribution of nucleoside-analogue reverse-transcriptaseinhibitor therapy to lipoatrophy from the population to thecellular level. Antiviral Therapy 2003; 8:617–626.

75. Cherry C, Nolan D, James I, Mallal S, McKinnon E,French M, Hammond E, Gahan M, McArthur J &Wesselingh S. Longitudinal associations between antiretro-viral treatments and quantification of tissue mitochondrialDNA from ambulatory subjects with HIV infection. 10thConference on Retroviruses & Opportunistic Infections.10–14 February 2003, Boston, Mass., USA. Abstract 133.

76. Hammond E, Nolan D, James I, Metcalf C & Mallal S.Reduction of mitochondrial DNA content and respiratorychain activity occurs in adipocytes within 6–12 months ofcommencing nucleoside reverse transcriptase inhibitortherapy. AIDS 2004; 18:563–565.

77. Staszewski S, Gallant JE, Pozniak AL, Suleiman JMAH,DeJesus E, Lu B, Sayre J & Cheng A. Efficacy and safety oftenofovir DF (TDF) versus stavudine (d4T) when used incombination with lamivudine and efavirenz in antiretro-viral naive patients: 96-week preliminary interim results.10th Conference on Retroviruses & OpportunisticInfections. 10–14 February 2003, Boston, Mass., USA.Abstract 564b.

78. Nolan D. Metabolic complications associated with HIVprotease inhibitor therapy. Drugs 2003; 63:2555–2574.

79. Keswani SC, Pardo CA, Cherry CL, Hoke A & McArthurJC. HIV-associated sensory neuropathies. AIDS 2002;16:2105–2117.

80. Berger AR, Arezzo JC, Schaumburg HH, Skowron G,Merigan T, Bozzette S, Richman D & Soo W. 2′,3′-dideoxycytidine (ddC) toxic neuropathy: a study of 52patients. Neurology 1993; 43:358–362.

81. Blum AS, Dal Pan GJ, Feinberg J, Raines C, Mayjo K,Cornblath DR & McArthur JC. Low-dose zalcitabine-related toxic neuropathy: frequency, natural history, andrisk factors. Neurology 1996; 46:999–1003.

82. Dalakas MC, Semino-Mora C & Leon-Monzon M.Mitochondrial alterations with mitochondrial DNA deple-tion in the nerves of AIDS patients with peripheralneuropathy induced by 2′3′-dideoxycytidine (ddC).Laboratory Investigation 2001; 81:1537–1544.

83. Moore RD, Wong WM, Keruly JC & McArthur JC.Incidence of neuropathy in HIV-infected patients onmonotherapy versus those on combination therapy withdidanosine, stavudine and hydroxyurea. AIDS 2000;14:273–278.

84. Simpson DM. Selected peripheral neuropathies associatedwith human immunodeficiency virus infection and anti-retroviral therapy. Journal of Neurovirology 2002; 8(Suppl. 2):33–41.

85. Simpson DM, Haidich AB, Schifitto G, Yiannoutsos CT,Geraci AP, McArthur JC & Katzenstein DA for the ACTG291 study team. Severity of HIV-associated neuropathy isassociated with plasma HIV-1 RNA levels. AIDS 2002;16:407–412.

86. Cherry C, McArthur J, Costello K, Woolley I, Mijch A &Wesselingh S. Increasing prevalence of neuropathy in theera of highly active antiretroviral therapy (HAART). 9thConference on Retroviruses & Opportunistic Infections.24–28 February 2002, Seattle, Wash., USA. Abstract 69.

87. Famularo G, Moretti S, Marcellini S, Trinchieri V,Tzantzoglou S, Santini G, Longo A & De Simone C.Acetyl-carnitine deficiency in AIDS patients with neurotox-icity on treatment with antiretroviral nucleoside analogues.AIDS 1997; 11:185–190.

88. Hart AM, Terenghi G, Johnson M, & Youle M.Immunohistochemical quantification of cutaneous innerva-tion in HIV-associated peripheral neuropathy: a study ofL-acetyl-carnitine therapy. Antiviral Therapy 2000;5(Suppl. 2):32. Abstract 36.

89. Moyle GJ & Sadler M. Peripheral neuropathy with nucleo-side antiretrovirals: risk factors, incidence andmanagement. Drug Safety 1998; 19:481–494.

Nolan & Mallal


90. Moore RD, Keruly JC & Chaisson RE. Incidence of pancre-atitis in HIV-infected patients receiving nucleoside reversetranscriptase inhibitor drugs. AIDS 2001; 15:617–620.

91. Routy JP, Smith GH, Blank DW & Gilfix BM.Plasmapheresis in the treatment of an acute pancreatitisdue to protease inhibitor-induced hypertriglyceridemia.Journal of Clinical Apheresis 2001; 16:157–159.

92. Perry RC, Cushing HE, Deeg MA & Prince MJ. Ritonavir,triglycerides, and pancreatitis. Clinical Infectious Diseases1999; 28:161–162.

93. Wilmink T & Frick TW. Drug-induced pancreatitis. DrugSafety 1996; 14:406–423.

94. Argiris A, Mathur-Wagh U, Wilets I & Mildvan D.Abnormalities of serum amylase and lipase in HIV-positivepatients. American Journal of Gastroenterology 1999;94:1248–1252.

95. Havlir DV, Gilbert PB, Bennett K, Collier AC, Hirsch MS,Tebas P, Adams EM, Wheat LJ, Goodwin D, Schnittman S,Holohan MK & Richman DD for the ACTG 5025 StudyGroup. Effects of treatment intensification with hydrox-yurea in HIV-infected patients with virologic suppression.AIDS 2001; 15:1379–1388.

96. Mallal S, Nolan D, Witt C, Masel G, Martin AM, MooreC, Sayer D, Castley A, Mamotte C, Maxwell D, James I &Christiansen FT. Association between presence of HLA-B*5701, HLA-DR7, and HLA-DQ3 and hypersensitivity toHIV-1 reverse-transcriptase inhibitor abacavir. Lancet2002; 359:727–732.

97. Fisher EJ, Chaloner K, Cohn DL, Grant LB, Alston B,Brosgart CL, Schmetter B, El-Sadr WM & Sampson J forthe Terry Beirn Community Programs for Clinical Researchon AIDS. The safety and efficacy of adefovir dipivoxil inpatients with advanced HIV disease: a randomized,placebo-controlled trial. AIDS 2001; 15:1695–1700.

98. Cihlar T, Ho ES, Lin DC & Mulato AS. Human renalorganic anion transporter 1 (hOAT1) and its role in thenephrotoxicity of antiviral nucleotide analogs. Nucleosides,Nucleotides & Nucleic Acids 2001; 20:641–648.

99. Coca S & Perazella MA. Rapid communication: acuterenal failure associated with tenofovir: evidence of drug-induced nephrotoxicity. American Journal of Medicine2002; 324:342–344.

100. Verhelst D, Monge M, Meynard JL, Fouqueray B,Mougenot B, Girard PM, Ronco P & Rossert J. Fanconisyndrome and renal failure induced by tenofovir: a firstcase report. American Journal of Kidney Diseases 2002;40:1331–1333.

101. Lichtenstein KA, Delaney KM, Armon C, Ward DJ,Moorman AC, Wood KC & Holmberg SD for the HIVOutpatient Study Investigators. Incidence of and riskfactors for lipoatrophy (abnormal fat loss) in ambulatoryHIV-1-infected patients. Journal of Acquired ImmuneDeficiency Syndromes 2003; 32:48–56.

102. Nolan D & Mallal S. Effects of sex and race on lipodys-trophy pathogenesis. Journal of HIV Therapy 2001;6:32–36.

103. Lafeuillade A, Hittinger G & Chadapaud S. Increasedmitochondrial toxicity with ribavirin in HIV/HCV coinfec-tion. Lancet 2001; 357:280–281.

104. Kakuda TN & Brinkman K. Mitochondrial toxic effectsand ribavirin. Lancet 2001; 357:1802–1803.

105. Salmon-Ceron D, Chauvelot-Moachon L, Abad S,Silbermann B & Sogni P. Mitochondrial toxic effects andribavirin. Lancet 2001; 357:1803–1804.

106. Hong Z & Cameron CE. Pleiotropic mechanisms ofribavirin antiviral activities. Progress in Drug Research2002; 59:41–69.

107. Inagaki H, Matsushima Y, Ohshima M & Kitagawa Y.Interferons suppress mitochondrial gene transcription bydepleting mitochondrial transcription factor A (mtTFA).Journal of Interferon & Cytokine Research 1997;17:263–269.

108. Hammond EL, Sayer D, Nolan D, Walker UA, Ronde A,Montaner JS, Cote HC, Gahan ME, Cherry CL,Wesselingh SL, Reiss P & Mallal S. Assessment of precisionand concordance of quantitative mitochondrial DNAassays: a collaborative international quality assurancestudy. Journal of Clinical Virology 2003; 27:97–110.

109. Gahan ME, Miller F, Lewin SR, Cherry CL, Hoy JF, MijchA, Rosenfeldt F & Wesselingh SL. Quantification of mito-chondrial DNA in peripheral blood mononuclear cells andsubcutaneous fat using real-time polymerase chain reac-tion. Journal of Clinical Virology 2001; 22:241–247.

110. Reiss P, Casula M, De Ronde A, Weverling GJ, Goudsmit J& Lange JM. Greater and more rapid depletion of mito-chondrial DNA in blood of patients treated with dual(zidovudine+didanosine or zidovudine+zalcitabine) vs.single (zidovudine) nucleoside reverse transcriptaseinhibitors. HIV Medicine 2004; 5:11–14.

111. Petit C, Mathez D, Barthelemy C, Leste-Lasserre T,Naviaux RK, Sonigo P & Leibowitch J. Quantitation ofblood lymphocyte mitochondrial DNA for the monitoringof antiretroviral drug-induced mitochondrial DNA deple-tion. Journal of Acquired Immune Deficiency Syndromes2003; 33:461–469.

112. Miura T, Goto M, Hosoya N, Odawara T, Kitamura Y,Nakamura T & Iwamoto A. Depletion of mitochondrialDNA in HIV-1-infected patients and its amelioration byantiretroviral therapy. Journal of Medical Virology 2003;70:497–505.

113. Brinkman K. Management of hyperlactatemia: no need forroutine lactate measurements. AIDS 2001; 15:795–797.

114. Mallon P, Unemori P, Bowen M, Miller J, WinterbothamM, Kelleher A, Williams K, Cooper D & Carr A.Nucleoside reverse transcriptase inhibitors decrease mito-chondrial and PPARgamma gene expression in adiposetissue after only two weeks in HIV-uninfected healthyadults. 11th Conference on Retroviruses & OpportunisticInfections. 8–11 February 2004, San Francisco, Calif.,USA. Abstract 76.



Received 25 March 2004, accepted 23 September 2004

antiviral therapy9:849–863 review complications associated

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