Borrelia burgdorferi Is a Poor Inducer of Gamma Interferon: Amplification Induced by Interleukin-12.

Laboratory diagnosis of Lyme borreliosis (LB) is mainly based on serology, which has limitations, particularly in the early stages of the disease. In recent years there have been conflicting reports concerning a new diagnostic tool using the cytokine interferon-gamma (IFN-γ). Previous studies have generally found low concentrations of IFN-γ in early LB infection. The goal of this study is to investigate IFN-γ regulation during early LB and provide insights into the host response to B. burgdorferi. We performed in vitro experiments with whole blood assays and peripheral blood mononuclear cells (PBMCs) of LB patients and healthy volunteers exposed to B. burgdorferi and evaluated the IFN-γ response using ELISA and related interindividual variation in IFN-γ production to the presence of single nucleotide polymorphisms. IFN-γ production of B. burgdorferi-exposed PBMCs and whole blood was amplified by the addition of interleukin-12 (IL-12) to the stimulation system. This effect was observed after 24 h of B. burgdorferi stimulation in both healthy individuals and LB patients. The effect was highly variable between individuals, but was significantly higher in LB patients 6 weeks since the start of antibiotic treatment compared to healthy individuals. IL-12 p40 and IL-18 mRNA were upregulated upon exposure to B. burgdorferi, whereas IL-12 p35 and IFN-γ mRNA expression remained relatively unchanged. SNP Rs280520 in the downstream IL-12 pathway, Tyrosine Kinase 2, was associated with increased IFN-γ production. This study shows that IL-12 evokes an IFN-γ response in B. burgdorferi exposed cells, and that LB patients and healthy controls respond differently to this stimulation.

Randomized Trial of Longer-Term Therapy for Symptoms Attributed to Lyme Disease.

BACKGROUND: The treatment of persistent symptoms attributed to Lyme disease remains controversial. We assessed whether longer-term antibiotic treatment of persistent symptoms attributed to Lyme disease leads to better outcomes than does shorter-term treatment. METHODS: In a randomized, double-blind, placebo-controlled trial conducted in Europe, we assigned patients with persistent symptoms attributed to Lyme disease--either related temporally to proven Lyme disease or accompanied by a positive IgG or IgM immunoblot assay for Borrelia burgdorferi--to receive a 12-week oral course of doxycycline, clarithromycin plus hydroxychloroquine, or placebo. All study groups received open-label intravenous ceftriaxone for 2 weeks before initiating the randomized regimen. The primary outcome measure was health-related quality of life, as assessed by the physical-component summary score of the RAND-36 Health Status Inventory (RAND SF-36) (range, 15 to 61, with higher scores indicating better quality of life), at the end of the treatment period at week 14, after the 2-week course of ceftriaxone and the 12-week course of the randomized study drug or placebo had been completed. RESULTS: Of the 281 patients who underwent randomization, 280 were included in the modified intention-to-treat analysis (86 patients in the doxycycline group, 96 in the clarithromycin-hydroxychloroquine group, and 98 in the placebo group). The SF-36 physical-component summary score did not differ significantly among the three study groups at the end of the treatment period, with mean scores of 35.0 (95% confidence interval [CI], 33.5 to 36.5) in the doxycycline group, 35.6 (95% CI, 34.2 to 37.1) in the clarithromycin-hydroxychloroquine group, and 34.8 (95% CI, 33.4 to 36.2) in the placebo group (P=0.69; a difference of 0.2 [95% CI, -2.4 to 2.8] in the doxycycline group vs. the placebo group and a difference of 0.9 [95% CI, -1.6 to 3.3] in the clarithromycin-hydroxychloroquine group vs. the placebo group); the score also did not differ significantly among the groups at subsequent study visits (P=0.35). In all study groups, the SF-36 physical-component summary score increased significantly from baseline to the end of the treatment period (P<0.001). The rates of adverse events were similar among the study groups. Four serious adverse events thought to be related to drug use occurred during the 2-week open-label ceftriaxone phase, and no serious drug-related adverse event occurred during the 12-week randomized phase. CONCLUSIONS: In patients with persistent symptoms attributed to Lyme disease, longer-term antibiotic treatment did not have additional beneficial effects on health-related quality of life beyond those with shorter-term treatment. (Funded by the Netherlands Organization for Health Research and Development ZonMw; PLEASE ClinicalTrials.gov number, NCT01207739.).

Cognitive impairments in patients with persistent symptoms attributed to Lyme disease.

BACKGROUND: Persistent symptoms attributed to Lyme borreliosis often include self-reported cognitive impairment. However, it remains unclear whether these symptoms can be substantiated by objective cognitive testing. METHODS: For this observational study, cognitive performance was assessed in 280 adults with persistent symptoms attributed to Lyme borreliosis (as part of baseline data collected for the Dutch PLEASE study). Cognitive testing covered the five major domains: episodic memory, working memory / attention, verbal fluency, information-processing speed and executive function. Patients' profiles of test scores were compared to a large age-, education- and sex-adjusted normative sample using multivariate normative comparison. Performance validity was assessed to detect suboptimal effort, and questionnaires were administered to measure self-reported cognitive complaints, fatigue, anxiety, depressive symptoms and several other psychological factors. RESULTS: Of 280 patients, one was excluded as the test battery could not be completed. Of the remaining 279 patients, 239 (85.4%) displayed sufficient performance validity. Patients with insufficient performance validity felt significantly more helpless and physically fatigued, and less orientated. Furthermore, they had a lower education level and less often paid work. Of the total study cohort 5.7% (n = 16) performed in the impaired range. Among the 239 patients who displayed sufficient performance validity, 2.9% (n = 7) were classified as cognitively impaired. No association between subjective cognitive symptoms and objective impairment was found. CONCLUSIONS: Only a small percentage of patients with borreliosis-attributed persistent symptoms have objective cognitive impairment. Performance validity should be taken into account in neuropsychological examinations of these patients. Self-report questionnaires are insufficiently valid to diagnose cognitive impairment. TRIAL REGISTRATION: ClinicalTrials.gov NCT01207739 . Registered 23 September 2010.

Persistent Lyme Empiric Antibiotic Study Europe (PLEASE)--design of a randomized controlled trial of prolonged antibiotic treatment in patients with persistent symptoms attributed to Lyme borreliosis.

BACKGROUND: Lyme borreliosis, a potentially severe tick-borne infection caused by Borrelia burgdorferi, can cause multi-system inflammatory disease. The incidence has been increasing, as has the number of patients with persistent symptoms attributed to Borrelia. These symptoms, also referred to as post-Lyme disease syndrome, may follow an erythema migrans or other Lyme manifestations, and include pain, fatigue, and cognitive disturbances. The optimal duration of treatment for these symptoms is a subject of controversy. The PLEASE study is designed to determine whether prolonged antibiotic treatment leads to better patient outcome than standard treatment. METHODS/DESIGN: The PLEASE study is a double-blind, randomized, placebo-controlled trial. Based on power analysis and compensating for possible loss to follow-up, a minimum of 255 patients with borreliosis-attributed persistent symptoms are included. These symptoms are either (a) temporally related to an erythema migrans or otherwise proven symptomatic borreliosis, or (b) accompanied by a positive B. burgdorferi IgG or IgM immunoblot. All patients receive open-label ceftriaxone for two weeks. Patients are then randomized (ratio 1:1:1) to blinded oral follow-up treatment for 12 weeks with (I) doxycycline, (II) clarithromycin combined with hydroxychloroquine, or (III) placebo. The primary outcome is the physical component summary score (PCS) of the RAND-36 Health Status Inventory (RAND SF-36) at week 14. Secondary outcomes include physical and mental aspects of health-related quality of life (assessed by the subscales of the RAND SF-36), fatigue, neuropsychological evaluation, physical activity, and cost-effectiveness. DISCUSSION: This article describes the background and design issues of the PLEASE study protocol. The results of this study may provide evidence for prescribing or withholding prolonged antibiotic treatment. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01207739 , Netherlands Trial Register: NTR2469.

Recognition of Borrelia burgdorferi by NOD2 is central for the induction of an inflammatory reaction.

Toll-like receptor 2 (TLR2) plays an important role in the recognition of Borrelia bacteria, the causative agent of Lyme disease, but the existence and importance of additional receptors in this process has been hypothesized. In the present study, we confirmed the role played by TLR2 in the recognition of Borrelia bacteria but also demonstrated a crucial role for the intracellular peptidoglycan receptor NOD2 for sensing the spirochete. Cells from individuals who were homozygous for the loss-of-function mutation 3020insC in the NOD2 gene were defective with respect to cytokine release after stimulation with Borrelia species, and this was confirmed in peritoneal macrophages from mice lacking RICK, the adaptor molecule used by NOD2. In contrast, NOD1 played no major role in the recognition of Borrelia spirochetes. This raises the intriguing possibility that recognition of Borrelia spirochetes is exerted by TLR2 in combination with NOD2 and that both receptors are necessary for an effective induction of cytokines by Borrelia species. The interplay between TLR2 and NOD2 might not only be necessary for the induction of a proper immune response but may also contribute to inflammatory-induced pathology.

Effect of prolonged antibiotic treatment on cognition in patients with Lyme borreliosis.

OBJECTIVE: To investigate whether longer-term antibiotic treatment improves cognitive performance in patients with persistent symptoms attributed to Lyme borreliosis. METHODS: Data were collected during the Persistent Lyme Empiric Antibiotic Study Europe (PLEASE) trial, a randomized, placebo-controlled study. Study participants passed performance-validity testing (measure for detecting suboptimal effort) and had persistent symptoms attributed to Lyme borreliosis. All patients received a 2-week open-label regimen of intravenous ceftriaxone before the 12-week blinded oral regimen (doxycycline, clarithromycin/hydroxychloroquine, or placebo). Cognitive performance was assessed at baseline and after 14, 26, and 40 weeks with neuropsychological tests covering the cognitive domains of episodic memory, attention/working memory, verbal fluency, speed of information processing, and executive function. RESULTS: Baseline characteristics of patients enrolled (n = 239) were comparable in all treatment groups. After 14 weeks, performance on none of the cognitive domains differed significantly between the treatment arms (p = 0.49-0.82). At follow-up, no additional treatment effect (p = 0.35-0.98) or difference between groups (p = 0.37-0.93) was found at any time point. Patients performed significantly better in several cognitive domains at weeks 14, 26, and 40 compared to baseline, but this was not specific to a treatment group. CONCLUSIONS: A 2-week treatment with ceftriaxone followed by a 12-week regimen of doxycycline or clarithromycin/hydroxychloroquine did not lead to better cognitive performance compared to a 2-week regimen of ceftriaxone in patients with Lyme disease-attributed persistent symptoms. CLINICALTRIALSGOV IDENTIFIER: NCT01207739. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that longer-term antibiotics in patients with borreliosis-attributed persistent symptoms does not increase cognitive performance compared to shorter-term antibiotics.

Stavudine plasma concentrations and lipoatrophy.

OBJECTIVES: The objective of this study was to determine the correlation between plasma stavudine concentrations and lipoatrophy (LA), one of the major adverse events in patients on stavudine and one of the major reasons to discontinue stavudine. METHODS: Plasma drug concentrations were retrospectively analysed in patients who were on a stavudine-containing regimen for at least 12 months. We defined two groups of patients: 21 patients with LA and 15 patients without LA or other stavudine-related side effects (i.e. neuropathy). RESULTS: We analysed stavudine concentrations in 212 plasma samples: 87 in the control group and 125 in the LA group, with a mean of four plasma samples per person (at least two a year). Demographics were comparable in LA patients and controls, except the duration of stavudine use, which was longer in the LA group: 55 versus 42 months in the control group. Overall, LA patients had higher drug exposure to stavudine when compared with the controls, and this was seen in the geometric concentration ratios (CRs), which were 0.978 and 0.741, respectively (P = 0.04), and also a higher percentage of CR values >1.0, representing a drug concentration above the normal population curve (46% versus 23%, P = 0.02). In addition, the duration of stavudine therapy was independently associated with LA (P = 0.05). In the multivariate analysis, both duration of stavudine (P = 0.05) and CR > 1.0 (P = 0.02) were independently correlated with LA. CONCLUSIONS: Monitoring of plasma stavudine concentrations can be useful to prevent stavudine-related LA.

Cost-effectiveness of longer-term versus shorter-term provision of antibiotics in patients with persistent symptoms attributed to Lyme disease.

BACKGROUND: The treatment of persistent symptoms attributed to Lyme disease remains controversial. Recently, the PLEASE study did not demonstrate any additional clinical benefit of longer-term versus shorter-term antibiotic treatment. However, the economic impact of the antibiotic strategies has not been investigated. METHODS: This prospective economic evaluation, adhering a societal perspective, was performed alongside the PLEASE study, a multicenter, placebo-controlled, double-blind 1:1:1 randomized clinical trial in which all patients received open-label intravenous ceftriaxone for two weeks before the 12-week randomized blinded oral antibiotic regimen (doxycycline, clarithromycin plus hydroxychloroquine, or placebo). Between 2010 and 2013, patients (n = 271) with borreliosis-attributed persistent symptoms were enrolled and followed for one year. Main outcomes were costs, quality-adjusted life years, and incremental net monetary benefit of longer-term versus shorter-term antibiotic therapy. RESULTS: Mean quality-adjusted life years (95% CI) were not significantly different (p = 0.96): 0.82 (0.77-0.88) for ceftriaxone/doxycycline (n = 82), 0.81 (0.76-0.88) for ceftriaxone/clarithromycin-hydroxychloroquine (n = 93), and 0.81 (0.76-0.86) for ceftriaxone/placebo (n = 96). Total societal costs per patient (95% CI) were not significantly different either (p = 0.35): €11,995 (€8,823-€15,670) for ceftriaxone/doxycycline, €12,202 (€9,572-€15,253) for ceftriaxone/clarithromycin-hydroxychloroquine, and €15,249 (€11,294-€19,781) for ceftriaxone/placebo. Incremental net monetary benefit (95% CI) for ceftriaxone/doxycycline compared to ceftriaxone/placebo varied from €3,317 (-€2,199-€8,998) to €4,285 (-€6,085-€14,524) over the willingness-to-pay range, and that of ceftriaxone/clarithromycin-hydroxychloroquine compared to ceftriaxone/placebo from €3,098 (-€888-€7,172) to €3,710 (-€4,254-€11,651). For every willingness-to-pay threshold, the incremental net monetary benefits did not significantly differ from zero. CONCLUSION: The longer-term treatments were similar with regard to costs, effectiveness and cost-effectiveness compared to shorter-term treatment in patients with borreliosis-attributed persistent symptoms after one year of follow-up. Given the results of this study, and taking into account the external costs associated with antibiotic resistance, the shorter-term treatment is the antibiotic regimen of first choice.

Lopinavir/ritonavir reduces lamotrigine plasma concentrations in healthy subjects.

BACKGROUND: Limited data are available about the effect of lopinavir and low-dose ritonavir on glucuronidation. Lamotrigine undergoes glucuronidation. We studied the effect of lopinavir/ritonavir on the pharmacokinetics of lamotrigine and vice versa. METHODS: Twenty-four healthy subjects received 50 mg lamotrigine once daily on days 1 and 2 and 100 mg twice daily on day 3 through day 23. Lopinavir (400 mg twice daily)/ritonavir (100 mg twice daily) was added on day 11. Depending on the decrease in lamotrigine trough level between days 10 and 20, either the study was stopped (<20% decrease) or a dose increase was applied from day 23 to day 31, as follows: increase to 150 mg lamotrigine twice daily if there was a 20% to 33% decrease, increase to 200 mg twice daily if there was a 34% to 66% decrease, and increase to 300 mg twice daily if there was a greater than 66% decrease. On days 10, 20, and 31, 12-hour pharmacokinetic curves were drawn. RESULTS: The mean decrease in lamotrigine trough level between days 10 and 20 was 55.4% (n = 18). A dose increment to 200 mg lamotrigine twice daily was used in all subjects. The area under the plasma concentration-time curve (AUC) values of lamotrigine on day 20 (with lopinavir/ritonavir) and day 10 (without lopinavir/ritonavir) were bioinequivalent, with a point estimate of 0.50 (90% confidence interval, 0.47-0.54). After dose adjustment of lamotrigine to 200 mg twice daily, the AUC on day 31 (n = 15) was bioequivalent to that on day 10, with a point estimate of 0.91 (90% confidence interval, 0.82-1.02). The median AUC ratios of lamotrigine 2N-glucuronide to lamotrigine on day 10 and day 20 were 0.57 (interquartile range, 0.39-0.75) and 1.12 (interquartile range, 0.87-1.31). Pharmacokinetic parameters for lopinavir/ritonavir were similar to historical controls. CONCLUSION: Lopinavir/ritonavir decreases the AUC of lamotrigine, probably by induction of glucuronidation. A dose increment to 200% of the initial lamotrigine dose is needed to achieve concentrations similar to those with lamotrigine alone. Lamotrigine does not appear to affect the pharmacokinetics of lopinavir/ritonavir.

Serum L-lactate and pyruvate in HIV-infected patients with and without presumed NRTI-related adverse events compared to healthy volunteers.

BACKGROUND: Nucleoside reverse transcriptase inhibitors (NRTIs) used in antiretroviral therapy may cause mitochondrial toxicity. Mitochondrial dysfunction leads to disturbance of the glucose metabolism, resulting in an accumulation of L-lactate (L) and pyruvate (P), with an enhanced L/P ratio. OBJECTIVES: We analysed lactate and pyruvate blood samples of patients of our outpatient department. Aim of the analysis was to detect preliminary mitochondrial toxicity in patients on antiretroviral nucleoside analogues, which might result in disturbances of L, P, L/P ratio, bicarbonate (Bic) or beta-hydroxybutyrate/aceto-acetate (beta-HB/AA) ratios. STUDY DESIGN: Blood samples of L, P, Bic, beta-HB and AA were analysed in four groups of subjects. The first group (A) consisted of patients with presumed NRTI-related adverse events (n=21), the second group (B) consisted of patients without adverse events (n=28), the third group (C) were HIV-infected patients without antiretroviral therapy (n=6) and the last group (D) were healthy controls (n=12). The mean duration of NRTI-treatment was 18 months (range 0-78 months). RESULTS: The mean lactate level in group A was 2319 micromol/l (S.D. +/-1231, median 1741 micromol/l), in group B 1257 micromol/l (S.D. +/-607, median 1087), Group C 1285 (S.D. +/-451, median 1245 micromol/l) and 951 micromol/l (S.D. +/-270, median 979) in the healthy controls. No significant differences in pyruvate, L/P, Bic and beta-HB/AA were seen in the four groups. The mean lactate level in patients on stavudine was 1980 micromol/l (S.D. +/-1197) versus 1051 micromol/l (S.D. +/-395, P=0.01) in patients on zidovudine. All patients with lactate values above 2700 micromol/l (eight) experienced adverse events. CONCLUSION: Lactate levels were higher in patients with presumed NRTI-related adverse events. Furthermore, HIV patients receiving a stavudine containing antiretroviral therapy had higher lactate values than patients without stavudine. Although routine lactate measurement in all patients on antiretroviral therapy is not recommended, lactate measurement might be useful for follow up of patients with presumed NRTI-related adverse events and in patients with lactate levels above 2500 micromol/l. These patients require extra surveillance to evaluate if discontinuation of the current antiretroviral therapy is needed.

Oral glucose loading for detection of mitochondrial toxicity during HAART in HIV-infected patients.

Nucleoside reverse transcriptase inhibitors used in antiretroviral therapy may cause mitochondrial toxicity. Mitochondrial dysfunction leads to disturbance of the glucose metabolism, resulting in an accumulation of L-lactate. We tested the hypothesis that an oral glucose tolerance test (OGTT) can be used to detect mitochondrial toxicity in patients on antiretroviral nucleoside analogues. An OGTT was performed in 30 subjects: 16 HIV-infected treated patients without adverse events (group 1) and 14 HIV-infected patients with adverse events related to nucleoside reverse transcriptase inhibitor-induced mitochondrial toxicity (group 2). Lactate was measured at baseline and 60 and 120 min after glucose loading. At all time points the lactate levels were higher in the adverse events group compared to the other group, with the highest levels of lactate at t = 60 min (mean 1912 micromol/L, SD +/- 609); mean lactates in the group without adverse events was 1429 micromol/L (SD +/- 464). When levels above the upper limit of normal of 1800 micromol/L were used as an indication for mitochondrial toxicity, the sensitivity and specificity were 57% and 81%, respectively. The area under the ROC curve was 0.75. For L-lactate levels > 2000 micromol/L the specificity was 90%. An OGTT with measurement of lactate at baseline and one hour after glucose loading can detect (occult) hyperlactataemia in patients with mitochondrial impairment. From our study we suggest to perform an OGTT as an additional test in patients with symptoms suspect for adverse events to discern mitochondrial toxicity.