Hyperlactataemia in HIV-infected patients: the role of NRTI-treatment

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1 Antiviral Therapy 7: Hyperlactataemia in HIV-infected patients: the role of NRTI-treatment Saskia ME Vrouenraets 1, Marco Treskes 2, Rosa M Regez 1, Nancy Troost 1, Yvo M Smulders 3, Hugo M Weigel 1, PH Jos Frissen 1 and Kees Brinkman 1 * 1 Department of Internal Medicine and 2 Department of Clinical Chemistry, Onze Lieve Vrouwe Gasthuis, Amsterdam, The Netherlands 3 Department of Internal Medicine, Academisch Ziekenhuis Vrije Universiteit, Amsterdam, The Netherlands *Corresponding author: Tel: ; Fax: ; K.Brinkman@olvg.nl Background: Long-term treatment with nucleoside reverse transcriptase inhibitors (NRTIs) can induce mitochondrial dysfunction, most severely represented by lactic acidosis. Diagnostic tests for mitochondrial dysfunction are lacking, although persistently elevated serum lactate might be a surrogate marker. Objectives: To determine the occurrence of hyperlactataemia in HIV-infected patients on NRTI-treatment and to evaluate the possible risk factors. Methods: Cross-sectional analysis of lactic-acid levels in asymptomatic HIV-infected patients. Hyperlactactaemia was considered mild if between mmol/l, serious if >5 mmol/l and lactic acidosis was defined as lactic acid levels >5 mmol/l with bicarbonate <20 mmol/l. Possible risk factors, such as current and preceding NRTI-treatment as well as treatment with non-nucleoside reverse transcriptase inhibitors or protease inhibitors and concurrent liver disease, were analysed. Results: Two hundred and twenty three asymptomatic HIV-infected patients were studied, including 174 patients (78%) on NRTI treatment, 12 patients (5%) treated without NRTIs and 37 patients (17%) not treated. Mild hyperlactataemia was found in 42 patients (19%), from whom 38/42 (90%) were NRTI-treated and the remaining patients (4/42, 10%) received no treatment (χ 2, P<0.05). The significant risk factors for hyperlactataemia in the univariate analysis were NRTI-treatment as a group (P=0.03) and elevated ALT (P=0.008). In multivariate analysis NRTI use (P=0.05) and ALT level (P=0.03) remained a significant determinant of hyperlactataemia. Among the different individual NRTIs, a stavudinecontaining (P=0.004) and a zalcitabine-containing (P=0.07) regimen were most notably associated with the development of hyperlactataemia, whereas for the combinations of NRTIs, such association was only found for stavudine/lamivudine (P=0.05). Conclusions: A correlation between hyperlactataemia and NRTI treatment was found, but the value of routine lactate measurement for individual treatment monitoring remains uncertain. Introduction Long-term treatment with antiretroviral medication can induce a prolonged suppression of HIV replication leading to improvement of the immune system, but carries also the risk of cumulative toxicity. One of these toxicities is mitochondrial toxicity, induced by nucleoside reverse transcriptase inhibitors (NRTIs) [1,2]. NRTIs inhibit the function of DNA polymerase γ, the enzyme responsible for the replication of mitochondrial DNA (mtdna). This inhibition can lead to depletion of mtdna and mtdna-encoded proteins, which will impair the oxidative phosphorylation of the cell in different functioning organs [2]. Mitochondrial dysfunction is believed to be the most important pathophysiological process in the development of most of the long-term adverse events of the NRTIs [1,2]. Although this is not definitely proven for symptoms such as pancreatitis, bone-marrow suppression, neuropathy and cardiomyopathy, this mechanism is clear for myopathy, hepatic steatosis and lactic acidosis. The role of mitochondrial dysfunction in lipodystrophy is still hypothetical [3]. Lactic acidosis is the most severe manifestation of mitochondrial toxicity and turns out to be fatal in many cases. Therefore, early recognition is essential. It is unclear whether this lactic acidosis is preceded by a period of hyperlactataemia and prospective studies about the prevalence of elevated lactate levels are lacking. Hyperlactataemia results either from increased production in peripheral tissue or from decreased clearance, which occurs mainly in the liver. An impaired clearance, for example, as a consequence of hepatic steatosis or chronic hepatitis B or C infection, 2002 International Medical Press /02/$

2 SME Vrouenraets et al. is probably the most important mechanism for elevation of lactic acid levels [4]. Studies about the factors that influence lactate are missing as well. The purpose of this study was to determine the occurrence of hyperlactataemia in HIV-infected patients on NRTI-treatment and to evaluate possible risk factors. Methods Patients A cross-sectional analysis of lactic acid levels in asymptomatic HIV-infected patients was performed. This analysis took place in a large teaching hospital, located in the centre of Amsterdam. This hospital gives care to a large and varied population of HIV-infected patients, who were infected with HIV in particular by homosexual contact or intravenous drug use. During the period October to December 1999, all ambulatory HIV-positive patients who visited the outpatient clinic for routine check-up were enrolled in this study. Measurements of lactate, bicarbonate and chloride were performed, in addition to routine laboratory analysis. Patient records were studied for current and preceding NRTI treatment, as well as for concurrent liver disease. Laboratory monitoring All laboratory analyses were performed on blood taken during routine blood sampling. No special precautions were taken, such as resting, fasting or collection of blood without a tourniquet. Lactate samples were collected by standard venipuncture technique in sodium fluoride/oxalate tubes (vacutainer, Becton & Dickinson, Leiden, The Netherlands). Plasma was separated within 15 min after venipuncture by centrifugation (10 min at 1500g). After centrifugation, lactate was determined by the lactate oxidase method on a Hitachi/modular P800 system (Roche Diagnostics, Almere, The Netherlands). In short, hydrogen peroxide is formed upon oxidation of lactate by the specific enzyme lactate oxidase. Hydrogen peroxide reacts with 4- aminoantipyrine to form a coloured dye with peroxidase as a catalyst. The increase in absorption at 660 nm is proportional to the amount of lactate in the plasma sample. The whole procedure after venipuncture was performed within 30 min, making transportation on ice, therefore, unnecessary. The plasma lactate concentration was considered normal below 2.0 mmol/l. Hyperlactataemia was defined as mild if lactate values were found from 2.1 to 5.0 mmol/l, and as serious for values above 5.0 mmol/l. Lactic acidosis was defined as serious hyperlactataemia with a bicarbonate level below 20 mmol/l. Liver enzymes (ALAT, ASAT) were analysed routinely for every patient, and status of hepatitis B and C infection was determined from the patient file. Statistical analysis Univariate analyses were performed to identify factors associated with hyperlactataemia, using the χ 2 test for nominal variables, and the independent sample t-test and Mann Whitney U-test for continuous variables. To assess an independent association between variables and hyperlactataemia, multiple logistic regression analysis was conducted using the Enter approach. We first evaluated different classes of drugs with respect to their possible effect on blood lactate levels. Subsequently, if a drug class was found to correlate with hyperlactataemia, a separate analysis of the individual drugs within this class was performed. A P-value of 0.05 or less was considered to indicate statistical significance. All data were analysed with SPSS statistical software (SPSS 9.0 for Windows). Results Of 223 HIV-infected patients, 174 patients (78%) were on a therapy regimen containing NRTIs. NRTI therapy, with or without protease inhibitor (PI) therapy, consisted mainly of zidovudine- (n=104) or stavudine- (n=56) containing regimens. All these regimens included combinations with either lamivudine, zalcitabine, didanosine or abacavir, or even a combination of two additional NRTIs. Concerning other antiretroviral therapy (ART), 54 patients (24%) received non-nucleoside reverse transcriptase inhibitors (NNRTIs) and 79 patients (35%) a regimen containing PIs. Thirty-seven patients (17%) did not receive ART. All were clinically asymptomatic (no severe abdominal pain, profuse vomiting or persistent nausea). Data for patients with and without hyperlactataemia are shown in Table 1. The lactic acid level was elevated in 42 patients (19%). Only one patient on a combination with NRTI and PI-treatment had serious hyperlactataemia, although without acidosis, which normalised when tested 2 days later. Retrospective questioning revealed that the initial sample (lactate level of 5.1 mmol/l) was collected shortly after the patient arrived at the hospital, having cycled. Variables positively associated with hyperlactataemia in univariate analyses included age, use of NRTI, ALT level and triglyceride levels. A trend was seen for lower CD4 counts and more hepatitis C virus positivity in hyperlactataemic subjects. The duration of current therapy was not a significant variable in our study (data not shown). Figure 1 shows a significant difference between the number of patients on NRTI-treatment with hyperlactataemia compared with patients with no or other International Medical Press

3 Hyperlactataemia in HIV-infected patients Table 1. Baseline characteristics Normal lactate Hyperlactatemia P (n=181) (n=42) Male sex no. (%) 160 (88) 37 (88) 0.96 Mean age years 39.6 (SD 7.7) 42.1 (SD 8.7) 0.06 Mean body mass index 23.2 (SD 3.1) 23.4 (SD 3.5) 0.84 weight/length 2 CD4 cell count/mm 3 median range Plasma HIV-1 RNA level complete suppression yes (%) 83 (46) 25 (60) 0.14 Median duration of antiretroviral therapy month (range) 14 ( ) 20.5 (1 90) 0.22 NRTI no. (%) 136 (75) 38 (90) 0.03 NNRTI no. (%) 54 (30) 15 (36) 0.46 PI no. (%) 79 (44) 20 (48) 0.64 No therapy no. (%) 33 (20) 4 (10) 0.17 Median ALT U/l (range) 16 (1-158) 21.5 (7 126) HbsAg-positive no. (%) 14 (8) 3 (7) 0.89 HCV-positive no. (%) 23 (13) 9 (22) 0.16 Median triglycerides mmol/l (range) 1.7 ( ) 2.2 ( ) 0.06 Data are represented as number (%), mean (standard deviation) or median (range). P-values are calculated by χ 2 test, unpaired Student t-test or Mann Whitney U-test. treatment regimens (Mann Whitney U-test, P=0.03). Based on the data in Table 1, age and ALT levels were considered to be the most important confounders of the association between NRTI use and hyperlactataemia, and were therefore included in the multiple logistic regression model (Table 2). Together with ALT levels, NRTI use remained a significant determinant of hyperlactataemia. Additional adjustment for triglyceride levels or CD4 count, or replacing ALT levels for the nominal variable hepatitis C virus positivity, did not materially affect these results (data not shown). In Table 3, the NRTI and NRTI combinations are analysed separately. Among the different NRTIs, stavudine (P=0.003) and zalcitabine (P=0.07) regimens appeared most notably associated with the development of hyperlactataemia, although the number of subjects in the zalcitabine group was relatively small for a reliable effect estimation. Among the NRTI combinations, especially the use of stavudine/lamivudine was a risk factor for lactate elevation. Adjustment of these individual drug correlations for age and ALT levels did not affect these results, and no other confounders were identified (data not shown). Within the other drug classes, no individual drug showed significant associations with increased blood lactate levels (data not shown). In 207 (37 not treated and 166 treated with NRTIs) patients, a second blood sample was drawn after 2 3 months. Regarding patients on NRTI treatment, lactate levels remained normal in 117/138 patients (85%), whereas mild hyperlactataemia returned to normal in 21/35 patients (60%) (data not shown). In only 14/35 patients (40%), mild hyperlactataemia persisted. In 16/133 patients (12%), a normal lactate level became mildly elevated. Discussion In this study, we found hyperlactataemia to occur significantly more frequently in patients on NRTI treatment compared with controls without NRTI treatment. Other investigators have studied the occurrence of hyperlactataemia during antiretroviral therapy as well [5 9]. In the studies by Boubaker et al. [7], Harris et al. [5] and Gérard et al. [9], untreated patients were not included in the analysis, making a conclusion Antiviral Therapy 7:4 241

4 SME Vrouenraets et al. about the role of NRTIs as a group impossible. John [10] and Moyle [8] included untreated patients: both found a lower occurrence of hyperlactataemia when no treatment was compared with any individual NRTI. Regarding the interpretation of elevated lactate levels, one has to consider that lactate measurement is susceptible to several confounding factors. Apart from the condition of the patient (preferably at rest), the sampling of blood is sensitive to artificial results. Use of a tourniquet and clenching of the fist for less than 1 min does not affect lactate levels significantly [11]. Preservation of the blood with sodium fluoride and oxalate and centrifugation within 30 min only results in a small increase of lactate (5 15%). The best way to produce reliable and reproducible results can be obtained by using either arterial or venous blood without long use of a tourniquet, from a patient at rest. Metabolism of lactate in the tube has to be prevented by putting the sample immediately on ice and/or by rapid processing. Furthermore, de novo lactate production from anaerobic glycolysis (in particular in the erythrocytes) needs to be interrupted by sampling in fluorinated tubes [12]. In the face of all these caveats, we still decided to evaluate lactate levels during routine blood sampling (with uncontrolled patient conditions and regular sampling of venous blood), to learn the value of this measurement in a clinical and not necessarily a research setting. Since most of the confounding factors discussed above might lead to artificially elevated levels, it was planned to repeat the measurement under standardised conditions in cases of serious hyperlactataemia; this Figure 1. Different distribution of lactate values for the NRTI versus the NNRTI group (P=0.03; Mann Whitney U-test) no NRTI 174 NRTI Median values (line) and percentiles (boxes) in both groups are shown. Table 2. Risk factors for hyperlactataemia (multivariate analysis) OR * 95% CI P Age (per 5 years) NRTI (yes) ALT (per 10 U/l) * odds ratio; 95% confidence interval. was only necessary on one occasion. Since we found the significant difference between the NRTI and the untreated group, we believe it to be highly unlikely that the hyperlactataemia observed resulted from artefacts in drawing and handling blood samples: both in controls and in treated patients the sampling was performed identically, and the laboratory technicians were blinded for treatments given. In this study the prevalence of asymptomatic hyperlactataemia in HIV-infected patients, including untreated patients, was 19%. Serious hyperlactataemia or lactic acidosis did not occur in these clinically asymptomatic individuals. We initially had one patient with serious hyperlactataemia (5.1 mmol/l), which turned out to be exercise-induced. Similar findings were reported by Harris et al. [5]: in a cross-sectional analysis of 70 NRTI-treated patients, 36% had a lactate above 2.1 mmol/l, while 13% had levels above 3.0 mmol/l. The fact that they observed a higher proportion of hyperlactataemia might be explained by the fact that almost all of their patients used stavudine (39% had a combination with hydroxyurea) (see below). Boubaker et al. [7] reported increased lactates (>1.1-times the upper normal limit) in 8.3% of their treated population, while Moyle et al. [8] found 8.7% of their patients to have a lactate >2.5 mmol/l. In none of these studies, the methodology of sampling was outlined in detail, which might possibly explain the differences observed. Evaluation of the risk factors associated with hyperlactataemia revealed NRTI treatment as a group as one of the major culprits. When analysing the NRTIs separately, there was an increased risk for hyperlactataemia with the use of stavudine or zalcitabine and when studying combinations of NRTIs, only stavudine/lamivudine appeared to be a significant risk factor. Also John et al. discerned the use of stavudine treatment as a significantly increased risk factor for developing higher lactate levels compared with both zidovudine or no treatment [10], as well as Harris et al. [5]. In a cross-sectional analysis of 880 patients in the Swiss cohort, again stavudine treatment, especially in combination with didanosine, was found to give an increased risk for elevated lactate compared with zidovudine [7]. However, when stavudine was studied without didanosine, the statistical significance of this finding got lost. Finally, in their cohort of International Medical Press

5 Hyperlactataemia in HIV-infected patients Table 3. The role of NRTI-treatment in hyperlactataemia (univariate analysis, not adjusted) n OR * CI P AZT d4t TC ddi ddc ABC AZT/3TC D4T/3TC D4T/ddI AZT/3TC/ABC * odds ratio; confidence interval. patients, Moyle et al. also found the highest prevalence of hyperlactataemia in stavudine/didanosine-treated patients (17.1%), but in their uni- and multivariate analysis, they conclude that especially current didanosine use is the most significant risk factor, whereas no difference in risk factors was found for current zidovudine or stavudine use [8]. In conclusion, both from our own study and from most other studies, stavudine use was the most suspicious risk factor for the development of hyperlactataemia, although the study of Moyle et al. in particular seems to discredit more the use of didanosine. Only prospective, randomised studies will definitely shed light on this issue. Since mitochondrial toxicity is considered to be a cumulative toxicity, we analysed whether time of NRTI exposure was a risk factor for increased lactate levels, but no statistical relation was found (data not shown). Furthermore, longitudinal follow-up with a second specimen per patient (n=201), revealed wide inter-individual fluctuation, while elevated lactates were also not consistent in the same individuals. The positive predictive value of asymptomatic hyperlactataemia found by routine measurement remains very low. Finally, we analysed whether concomitant liver disease was an increased risk factor for hyperlactataemia, since the liver forms the most important organ for clearance of lactate from the circulation [4]. In fact, in small studies on serious hyperlactataemia and lactic acidosis, liver involvement was found in all cases studied [13 22]. In most of these cases, profound hepatic steatosis was found, which is believed to be a serious feature of impaired mitochondrial dysfunction [23]. In our study, a statistically significant correlation between lactate levels and ALT was found but no correlation between hyperlactataemia and positive hepatitis serology (Table 1). A similar finding was observed in the study of Moyle et al. [8], but not in the studies of John et al. [10] and Boubaker et al. [7]. The question, whether hyperlactataemia is more a reflection of impaired liver clearance than of increased production in for instance adipose tissue as suggested by Carr et al. [24], remains unresolved. Analysing both our results and the results presented in the literature, one can conclude that mild hyperlactataemia occurs relatively frequent (8.2 36%) in NRTI-treated patients, depending on the NRTI treatment used. The clinical meaning of such a persistent, but mild, elevated lactate level remains uncertain, since prospective data on its consequences are lacking. However, serious hyperlactataemia (>5.0 mmol/l) was hardly observed in asymptomatic patients in any of the studies. Based on these findings, we would propose that no change in treatment should be advised in cases of asymptomatic, mild hyperlactataemia (2.0 mmol/l < lactate < 5.0 mmol/l), only a close follow-up, with special emphasis on other signs of mitochondrial toxicity. In cases of serious hyperlactataemia (>5.0 mmol/l), treatment alterations should be considered. Future, prospective studies need to be performed to confirm such guidelines [25]. As long as these studies have not been performed, one should be cautious in implementing lactate measurements in routine clinical practice. Acknowledgements Dr GJ Weverling is acknowledged for useful statistical advice. References 1. Lewis W & Dalakas MC. 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