An Enzyme-Linked Immunosorbent Spot Assay Measuring Borrelia burgdorferi B31-Specific Interferon Gamma-Secreting T Cells Cannot Discriminate Active Lyme Neuroborreliosis from Past Lyme Borreliosis: a Prospective Study in the Netherlands.

Two-tier serology testing is most frequently used for the diagnosis of Lyme borreliosis (LB); however, a positive result is no proof of active disease. To establish a diagnosis of active LB, better diagnostics are needed. Tests investigating the cellular immune system are available, but studies evaluating the utility of these tests on well-defined patient populations are lacking. Therefore, we investigated the utility of an enzyme-linked immunosorbent spot (ELISpot) assay to diagnose active Lyme neuroborreliosis. Peripheral blood mononuclear cells (PBMCs) of various study groups were stimulated by using Borrelia burgdorferi strain B31 and various recombinant antigens, and subsequently, the number of Borrelia-specific interferon gamma (IFN-γ)-secreting T cells was measured. We included 33 active and 37 treated Lyme neuroborreliosis patients, 28 healthy individuals treated for an early manifestation of LB in the past, and 145 untreated healthy individuals. The median numbers of B. burgdorferi B31-specific IFN-γ-secreting T cells/2.5 × 105 PBMCs did not differ between active Lyme neuroborreliosis patients (6.0; interquartile range [IQR], 0.5 to 14.0), treated Lyme neuroborreliosis patients (4.5; IQR, 2.0 to 18.6), and treated healthy individuals (7.4; IQR, 2.3 to 14.9) (P = 1.000); however, the median number of B. burgdorferi B31-specific IFN-γ-secreting T cells/2.5 × 105 PBMCs among untreated healthy individuals was lower (2.0; IQR, 0.5 to 3.9) (P ≤ 0.016). We conclude that the Borrelia ELISpot assay, measuring the number of B. burgdorferi B31-specific IFN-γ-secreting T cells/2.5 × 105 PBMCs, correlates with exposure to the Borrelia bacterium but cannot be used for the diagnosis of active Lyme neuroborreliosis

Disagreement between the results from three commercial tests for the detection of Borrelia-specific serum antibodies in the Netherlands associated with antibiotic treatment for Lyme borreliosis: a retrospective study.

The diagnosis of Lyme borreliosis is challenging because of the often non-specific symptoms and persisting antibodies after infection. We investigated the diagnostic characteristics of two enzyme-linked immunosorbent assays (ELISAs) and an immunoblot for the detection of Borrelia-specific serum antibodies using different test strategies in individuals with and without antibiotic treatment for Lyme borreliosis. This retrospective study included healthy individuals, patients with active Lyme neuroborreliosis and patients treated for Lyme neuroborreliosis. Two ELISAs were compared: the C6 ELISA and the SERION ELISA. Equivocal and positive results were confirmed by immunoblot. We included 174 healthy individuals, of whom 27 (15.5%) were treated for Lyme borreliosis in the past, 36 patients were treated for Lyme neuroborreliosis and 27 patients had active Lyme neuroborreliosis. All the active Lyme neuroborreliosis patients were reactive in both ELISAs (100% sensitivity); less reactivity was seen in the other three groups (range 17.7% to 69.4%). The concordance between the ELISA results was high in active Lyme neuroborreliosis patients (26/27; 96.3%) and healthy individuals (131/147; 89.1%), but lower in treated healthy individuals (18/27; 66.7%) and treated Lyme neuroborreliosis patients (18/36; 50.0%) (p ≤ 0.005). This study showed that antibiotic treatment against Lyme borreliosis was strongly associated with discordant ELISA and test strategy results (odds ratio: 10.52; p < 0.001 and 9.98; p = 0.014, respectively) suggesting antibiotic treatment influences the pace at which the various antibodies directed to the different antigens used in both ELISAs wane. Among treated neuroborreliosis patients, the SERION ELISA stayed positive for a longer period after infection compared to the C6 ELISA. This should be taken into consideration when requesting and/or interpreting Lyme serology

An “Escape Clock” for Estimating the Turnover of SIV DNA in Resting CD4+ T Cells

Persistence of HIV DNA presents a major barrier to the complete control of HIV infection under current therapies. Most studies suggest that cells with latently integrated HIV decay very slowly under therapy. However, it is much more difficult to study the turnover and persistence of HIV DNA during active infection. We have developed an “escape clock” approach for measuring the turnover of HIV DNA in resting CD4+ T cells. This approach studies the replacement of wild-type (WT) SIV DNA present in early infection by CTL escape mutant (EM) strains during later infection. Using a strain-specific real time PCR assay, we quantified the relative amounts of WT and EM strains in plasma SIV RNA and cellular SIV DNA. Thus we can track the formation and turnover of SIV DNA in sorted resting CD4+ T cells. We studied serial plasma and PBMC samples from 20 SIV-infected Mane-A*10 positive pigtail macaques that have a signature Gag CTL escape mutation. In animals with low viral load, WT virus laid down early in infection is extremely stable, and the decay of this WT species is very slow, consistent with findings in subjects on anti-retroviral medications. However, during active, high level infection, most SIV DNA in resting cells was turning over rapidly, suggesting a large pool of short-lived DNA produced by recent infection events. Our results suggest that, in order to reduce the formation of a stable population of SIV DNA, it will be important either to intervene very early or intervene during active replication

High frequency of APOB gene mutations causing familial hypobetalipoproteinaemia in patients of Dutch and Spanish descent

Background: Familial hypobetalipoproteinaemia (FHBL) is an autosomal co-dominant hereditary disorder of lipoprotein metabolism characterised by decreased low density lipoprotein (LDL) cholesterol and apolipoprotein B (APOB) plasma levels. High levels of plasma APOB and LDL cholesterol are strong predictors for risk of cardiovascular disease (CVD), while individuals with low APOB and LDL cholesterol levels are thought to have lower than average risk for CVD, and in fact, heterozygous FHBL patients appear to be asymptomatic. Methods: Rather than identifying truncated APOB proteins in plasma fractions separated by gel electrophoresis, which will miss any mutations in proteins smaller than 30 kb, we analysed the APOB gene directly, using PCR. Results: We identified nine different mutations, six of which are novel. Each mutation showed complete co-segregation with the FHBL phenotype in the families, and statistically significant differences between carriers and non-carriers were found for plasma total, LDL, and HDL cholesteroll, triglycerides, and APOB levels, but not for APOA1 levels. All carriers of an APOB mutation were completely free from CVD. Conclusions: Prolonged low levels of LDL cholesterol and elevated levels of HDL cholesterol may reduce the progression of atherosclerotic disease, but this has not been unequivocally shown that this is indeed the case in individuals with FHBL, and is the subject of a current study

Limited penetration of lopinavir into seminal plasma of HIV-1-infected men.

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Population pharmacokinetics of indinavir alone and in combination with ritonavir in HIV-1-infected patients

Aims The aim of the study was to characterize the population pharmacokinetics of indinavir, define the relationship between the pharmacokinetics of indinavir and ritonavir, and to identify the factors influencing the pharmacokinetics of indinavir alone or when given with ritonavir. Methods HIV-1-infected patients being treated with an indinavir-containing regimen were included. During regular visits, 102 blood samples were collected for the determination of plasma indinavir and ritonavir concentrations. Full pharmacokinetic curves were available from 45 patients. Concentrations of indinavir and ritonavir were determined by liquid chromatography coupled with electrospray tandem mass spectrometry. Pharmacokinetic analysis was performed using nonlinear mixed effect modelling (NONMEM). Results The disposition of indinavir was best described by a single compartment model with first order absorption and elimination. Values for the clearance, volume of distribution and the absorption rate constant were 46.8 l h(-1) (24.2% IIV), 82.3 l (24.6% IIV) and 02.62 h(-1), respectively. An absorption lag-time of 0.485 h was detected in patients also taking ritonavir. Furthermore this drug, independent of dose (100-400 mg) or plasma concentration, decreased the clearance of indinavir by 64.6%. In contrast, co-administration of efavirenz or nevirapine increased the clearance of indinavir by 41%, irrespective of the presence or absence of ritonavir. Female patients had a 48% higher apparent bioavailability of indinavir than males. Conclusions The pharmacokinetic parameters of indinavir were adequately described by our population model. Female gender and concomitant use of ritonavir and non-nucleoside reverse transcriptase inhibitors strongly influenced the pharmacokinetics of this drug. The results support the concept of ritonavir boosting, maximum inhibition of indinavir metabolized being observed at 100 m