Loop-mediated isothermal amplification for paratyphoid fever - a proof-of-principle analysis.

In-house loop-mediated isothermal amplification (LAMP) procedures for the detection of paratyphoid fever-associated bacteria on serovar level were evaluated. Therefore, LAMP primers for Salmonella genus, for two LAMP schemes for S. Paratyphi A, for S. Paratyphi B and for S. Paratyphi C were tested with DNA from culture isolates from strain collections and spiked blood cultures against published PCR protocols targeting the same micro-organisms. Sensitivity and specificity for DNA from culture isolates verified by LAMP ranged from 80·0 to 100·0% and 96·1 to 100·0% vs 65 to 100% and 98·7 to 100% for the PCR approaches. For the spiked blood culture materials, sensitivity and specificity for LAMP ranged from 87·5 to 100·0% and 96·7 to 100·0% vs from 60 to 100% and 98·2 to 100% for PCR. In conclusion, LAMP for paratyphoid fever shows comparable performance characteristics as PCR. Due to its easy application, the procedure is well suited for surveillance purposes in resource-limited settings. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of easy-to-apply, point-of-care-testing-like loop-mediated isothermal amplification (LAMP) for the diagnosis of paratyphoid fever is evaluated. This approach can contribute to low-threshold availability of surveillance options for resource limited settings. Easy-to-teach and easy-to-apply LAMP schemes with similar performance characteristics as PCR are provided. The described test evaluation is of particular use for surveillance and public health experts.

HIV prevention strategies and risk of infection: a model-based analysis.

Risk populations for HIV infections tend to neglect condom use, making alternative preventive approaches necessary. Accordingly, we modelled the risk of sexual HIV transmission for condom use vs. use of rapid diagnostic test (RDT) systems with subsequent exclusion of potential sexual partners with a correctly or falsely positive test from unprotected sex with and without the use of HIV pre-exposure prophylaxis (PrEP) in a bio-statistical approach. We combined a previously described model of transmission risk for HIV-exposed individuals with a newly suggested model of risk of HIV exposure for sexually active HIV-negative individuals. The model was adapted for several stages of infection and different strategies of HIV infection prevention.HIV prevention with RDTs can reduce the transmission risk by up to 97% compared with having sex without any prevention and up to 80% compared with condom use. Nevertheless, RDT-based prevention strategies demonstrate a lack of protection in several stages of infection; in particular, RNA-based RDT systems may fail under treatment. RDT-based pre-screening of potential sex partners prior to unprotected sexual contacts substantially reduces HIV transmission risk. Combination of different prevention strategies is advisable for high-risk groups.

In vivo imaging of brain microglial activity in antipsychotic-free and medicated schizophrenia: a [11C](R)-PK11195 positron emission tomography study.

Positron emission tomography (PET) imaging of the 18 kDa translocator protein (TSPO) has been used to investigate whether microglial activation, an indication of neuroinflammation, is evident in the brain of adults with schizophrenia. Interpretation of these studies is confounded by potential modulatory effects of antipsychotic medication on microglial activity. In the first such study in antipsychotic-free schizophrenia, we have used [11C](R)-PK11195 PET to compare TSPO availability in a predominantly antipsychotic-naive group of moderate-to-severely symptomatic unmedicated patients (n=8), similarly symptomatic medicated patients with schizophrenia taking risperidone or paliperidone by regular intramuscular injection (n=8), and healthy comparison subjects (n=16). We found no evidence for increased TSPO availability in antipsychotic-free patients compared with healthy controls (mean difference 4%, P=0.981). However, TSPO availability was significantly elevated in medicated patients (mean increase 88%, P=0.032) across prefrontal (dorsolateral, ventrolateral, orbital), anterior cingulate and parietal cortical regions. In the patients, TSPO availability was also strongly correlated with negative symptoms measured using the Positive and Negative Syndrome Scale across all the brain regions investigated (r=0.651-0.741). We conclude that the pathophysiology of schizophrenia is not associated with microglial activation in the 2-6 year period following diagnosis. The elevation in the medicated patients may be a direct effect of the antipsychotic, although this study cannot exclude treatment resistance and/or longer illness duration as potential explanations. It also remains to be determined whether it is present only in a subset of patients, represents a pro- or anti-inflammatory state, its association with primary negative symptoms, and whether there are significant differences between antipsychotics.

The effect of 18F-florbetapir dose reduction on region-based classification of cortical amyloid deposition.

UNLABELLED: There are specific dose recommendations for diagnostic amyloid PET imaging with 18F-florbetapir, but they may not apply to research studies using regional quantitative analysis. We, therefore, studied the effect of tracer dose reduction on the discriminative power of regional analysis. METHODS: Using bootstrap resampling of list-mode data from 18F-florbetapir scans, a total of 800 images were reconstructed for four different dosage levels: 100, 50, 20, and 10%. The effect of the injected dose on the variation of measured radiotracer uptake was determined in large cortical regions defined on co-registered and segmented magnetic resonance images. The impact of the observed variation on the discrimination between normal controls and patients with AD was then assessed using data in a cohort study described by Fleisher et al. (Arch Neurol 68(11):1404-1411, 2011). RESULTS: The coefficient of variance for the cortex to cerebellum uptake ratio increased from 0.9% at full dose of 300 MBq to 2.5% at 10% of this dose, but was still small compared to biological variation. It, therefore, had very little impact on discrimination between AD and elderly controls. The original area under the ROC curve was 0.881, decreasing to 0.878 at 10% of full dose. Original sensitivity for discrimination between AD and controls was 82.0%, while specificity was 77.3%; these decreased to 81.8 and 77.1%, respectively, at the reduced dose. However, the number of subjects within the classification border zone between proven amyloid pathology and young healthy controls increased substantially by 7 to 14%. CONCLUSION: A substantial reduction of tracer dose increases uncertainty at the classification border zone while still providing good discrimination between AD patients and controls when using activity data from cortical regions defined on co-registered and segmented MR scans.

[Endometrioid borderline tumor of the testis. A rare cause of cystic neoplasia]. / Endometrioider Borderlinetumor des Hodens. Eine seltene Ursache für eine zystische Raumforderung.

Ovarian type surface epithelial carcinomas of the testis are rare and therefore mostly represent a surprising finding in diagnostic procedures. The most frequent is the serous subtype, while only a few cases of the endometrioid subtype have been reported in the literature. The case of a 73-year-old patient with an endometrioid type papillary cystic tumor of borderline malignancy is presented. The histopathological and immunohistochemical details of this rare tumor are discussed.

Comparison of MRI based and PET template based approaches in the quantitative analysis of amyloid imaging with PIB-PET.

RATIONALE: [(11)C]Pittsburgh compound-B (PIB) has been the most widely used positron emission tomography (PET) imaging agent for brain amyloid. Several longitudinal studies evaluating the progression of Alzheimer's disease (AD), and numerous therapeutic intervention studies are underway using [(11)C]PIB PET as an AD biomarker. Quantitative analysis of [(11)C]PIB data requires the definition of regional volumes of interest. This investigation systematically compared two data analysis routes both using a probabilistic brain atlas with 11 bilateral regions. Route 1 used individually segmented structural magnetic resonance images (MRI) for each subject while Route 2 used a standardised [(11)C]PIB PET template. METHODS: A total of 54 subjects, 20 with probable Alzheimer's disease (AD), 14 with amnestic Mild Cognitive Impairment (MCI) and 20 age-matched healthy controls, were scanned at two imaging centres either in London (UK) or in Turku (Finland). For all subjects structural volumetric MRI and [(11)C]PIB PET scans were acquired. Target-to-cerebellum ratios 40 min to 60 min post injection were used as outcome measures. Regional read outs for grey matter target regions were generated for both routes. Based on a composite neocortical, frontal, posterior cingulate, combined posterior cingulate and frontal cortical regions, scans were categorised into either 'PIB negative' (PIB-) or 'PIB positive' (PIB+) using previously reported cut-off target-to-cerebellar ratios of 1.41, 1.5 and 1.6, respectively. RESULTS: Target-to-cerebellum ratios were greater when defined with a [(11)C]PIB PET template than with individual MRIs for all cortical regions regardless of diagnosis. This difference was highly significant for controls (p<0.001, paired samples t-test), less significant for MCIs and borderline for ADs. Assignment of subjects to raised or normal categories was the same with both routes with a 1.6 cut-off while with lower cut off using frontal cortex, and combined frontal cortex and posterior cingulate demonstrated similar results, while posterior cingulate alone demonstrated significantly higher proportion of controls as amyloid positive by Route 2. CONCLUSIONS: Definition of cortical grey matter regions is more accurate when individually segmented MRIs (Route 1) were used rather than a population-based PET template (Route 2). The impact of this difference depends on the grey-to-white matter contrast in the PET images; specifically seen in healthy controls with high white matter and low grey matter uptake. When classifying AD, MCI and control subjects as normal or abnormal using large cortical regions; discordance was found between the MRI and template approach for those few subjects who presented with cortex-to-cerebellum ratios very close to the pre-assigned cut-off. However, posterior cingulate alone demonstrated significant discordance in healthy controls using template based approach. This study, therefore, demonstrates that the use of a [(11)C]PIB PET template (Route 2) is adequate for clinical diagnostic purposes, while MRI based analysis (Route 1) remains more appropriate for clinical research.

Measurement of Protein Synthesis Rate in Rat by [11C]Leucine PET Imaging: Application to the TgF344-AD Model of Alzheimer's Disease.

Long-term memory requires stable protein synthesis and is altered in Alzheimer's disease (AD). This study aimed to implement a method to measure the cerebral protein synthesis rate (PSR) with [11C]leucine PET in vivo in rats and evaluate potential PSR alterations longitudinally (6, 12 and 18 months old) in the TgF344-AD rat model of AD. Wistar, wild-type (WT) and TgF344-AD rats (TG) were scanned for 60 min with [11C]leucine. Arterial blood activity was monitored online and with discrete whole blood and plasma samples by γ-counting in Wistar rats, WT (n = 4) and TG (n = 5). Unlabelled amino acids were measured in plasma. The sensitivity of [11C]leucine PET to measure alterations in PSR was assessed in Wistar rats by injection of PSR inhibitor anisomycin before PET acquisition. Anisomycin administration significantly reduced the net uptake rate constant (Kcplx) of [11C]leucine and PSR, proving the suitability of the method. For the longitudinal study, averaged population-based input functions were used to calculate PSR. We found a significant genotype effect on PSR (decrease in TG vs WT) only in the globus pallidus. This study suggests that [11C]leucine PET is sensitive enough to measure brain PSR in rat but that cross-sectional design with individual input function should be preferred.

Can target-to-pons ratio be used as a reliable method for the analysis of [11C]PIB brain scans?

RATIONALE: (11)C]PIB is the most widely used PET imaging marker for amyloid in dementia studies. In the majority of studies the cerebellum has been used as a reference region. However, cerebellar amyloid may be present in genetic Alzheimer's (AD), cerebral amyloid angiopathy and prion diseases. Therefore, we investigated whether the pons could be used as an alternative reference region for the analysis of [(11)C]PIB binding in AD. The aims of the study were to: 1) Evaluate the pons as a reference region using arterial plasma input function and Logan graphical analysis of binding. 2) Assess the power of target-to-pons ratios to discriminate controls from AD subjects. 3) Determine the test-retest reliability in AD subjects. 4) Demonstrate the application of target-to-pons ratio in subjects with elevated cerebellar [(11)C]PIB binding. METHODS: 12 sporadic AD subjects aged 65 ± 4.5 yrs with a mean MMSE 21.4 ± 4 and 10 age-matched control subjects had [(11)C]PIB PET with arterial blood sampling. Three additional subjects (two subjects with pre-symptomatic presenilin-1 mutation carriers and one probable familial AD) were also studied. Object maps were created by segmenting individual MRIs and spatially transforming the gray matter images into standard stereotaxic MNI space and then superimposing a probabilistic atlas. Cortical [(11)C]PIB binding was assessed with an ROI (region of interest) analysis. Parametric maps of the volume of distribution (V(T)) were generated with Logan analysis. Additionally, parametric maps of the 60-90 min target-to-cerebellar ratio (RATIO(CER)) and the 60-90 min target-to-pons ratio (RATIO(PONS)) were computed. RESULTS: All three approaches were able to differentiate AD from controls (p<0.0001, nonparametric Wilcoxon rank sum test) in the target regions with RATIO(CER) and RATIO(PONS) differences higher than V(T) with use of an arterial input function. All methods had a good reproducibility (intraclass correlation coefficient>0.83); RATIO(CER) performed best closely followed by RATIO(PONS). The two subjects with presenilin-1 mutations and the probable familial AD case showed no significant differences in cortical binding using RATIO(CER), but the RATIO(PONS) approach revealed higher [(11)C]PIB binding in cortex and cerebellum. CONCLUSION: This study established 60-90 min target-to-pons RATIOs as a reliable method of analysis in [(11)C]PIB PET studies where cerebellum is not an appropriate reference region.

In vitro percutaneous penetration: evaluation of the utility of hairless mouse skin.

The permeability barrier of hairless mouse skin has been determined in vitro after exposure of the epidermal surface to volumes of acetone typically used in human in vivo skin penetration studies. It has been shown that the transport of tritiated water (when applied for limited 5-h periods) across hairless mouse skin is not affected by acetone treatments of approximately 15 microliters/cm2. Submersion of the membranes between aqueous donor and receptor phases for periods greater than 24 h, however, leads to significant and catastrophic barrier impairment. The acetone dose in the experiments reported is greater than that employed in vivo when the solvent is used to deposit a penetrant on human skin. We suggest, therefore, that acetone-mediated facilitation of percutaneous absorption in humans is unlikely. A further conclusion of this work is that in vitro solvent-deposition penetration experiments using hairless mouse skin should provide reliable transport information for at least 48 h postadministration. Although hairless mouse skin is more permeable than its human counterpart, in vitro measurements using the murine barrier should, therefore, provide useful and relevant guidelines for risk assessment calculations and bioavailability determinations.

In vivo measurement of the serotonin transporter with (S)-([18F]fluoromethyl)-(+)-McN5652.

The radiolabeled serotonin transporter (SERT) ligand [(11)C](+)-McN5652 has recently been used in clinical positron emission tomography (PET) studies for SERT imaging. However, this radioligand offers disadvantages in routine clinical settings because of its short radioisotope half-life (eg PET facilities within hospitals without a cyclotron need to acquire such radioligands from distant cyclotron units for clinical use). S-([(18)F]fluoromethyl)-(+)-McN5652 ([(18)F](+)-FMe-McN5652) is an analogue which has been synthesized newly, and has a significantly longer radioisotope half-life. In the porcine brain, it demonstrates the same characteristic distribution pattern of serotonin-uptake sites like the (11)C-labeled congener with the highest binding in the midbrain and thalamus and the lowest in the cerebellum and occipital cortex. It shows a 30% higher blood-brain transfer and a slower peripheral metabolism than [(11)C](+)-McN5652. Rather uniform brain binding was observed after injection of the pharmacologically inactive radiolabeled enantiomer, or after pretreatment with the highly selective SERT inhibitor citalopram. The norepinephrine uptake inhibitor maprotiline did not show any inhibitory effect. Using a one-tissue compartment model (K(1), k"(2)) or a two-tissue compartment model (K(1) to k(4)) with or without constraints for calculation, the regional binding parameters of [(11)C](+)-McN5652 and [(18)F](+)-FMe-McN5652 are highly correlated among each other and with the SERT density, as determined by in vitro binding of [(3)H]citalopram. Using constraints to correct for the free fraction and nonspecific binding of the radiotracers, a considerable increase of the midbrain-occipital cortex ratios with higher values for [(18)F](+)-FMe-McN5652 compared to [(11)C](+)McN5652 was revealed. It is concluded that [(18)F](+)-FMe-McN5652 has better features than [(11)C](+)McN5652 for SERT imaging with PET.

Blockage of skin invasion by schistosome cercariae by serine protease inhibitors.

Invasion of skin by schistosome cercariae is facilitated by a serine protease secreted from the acetabular cells of cercariae in response to skin lipid. Specific inhibitors of the protease, when applied to human skin in formulations designed to retain the inhibitor on and in the upper stratum corneum layers, block cercarial invasion of human skin. Both peptide-based, irreversible inhibitors and non-peptide, reversible inhibitors block cercarial invasion when applied in a propylene glycol:isopropyl alcohol (3:1) formulation in vitro. Arrest of cercarial invasion could be achieved even after immersion of treated skin in water for 2 hr. Peptide-based irreversible inhibitors in the presence of three different Topicare Delivery Compounds optimized arrest of cercarial invasion. The three Topicare Delivery Compounds applied alone prevented 80-100% of cercarial invasion. With inclusion of the inhibitor, there was 97-100% inhibition in vitro. The optimal formulation with inhibitor was then applied to the tails of BALB/c mice, and the mice were exposed to 120 cercariae by tail immersion. With the carrier lotion alone, there was a 50% reduction in worm burden and a 70% reduction in egg burden. When inhibitor was included, an 80% reduction in worm burden and a 92% reduction in egg burden was observed.

Percutaneous penetration of nicotinates: in vivo and in vitro measurements.

The relationship between chemical structure and percutaneous absorption has been explored with nicotinic acid and its methyl, ethyl, hexyl, and benzyl esters. Skin penetration has been measured in vitro across hairless mouse skin and in vivo in humans. In vitro, methyl and ethyl nicotinates (when applied in acetone) were delivered into skin such that the stratum corneum barrier was effectively bypassed. The lipophilic esters, on the other hand, were not solubilized in this way and penetrated more slowly. Nicotinic acid penetrated poorly, yielding essentially zero-order skin transport kinetics. Tape-stripping experiments, in which penetration was monitored across skin with no stratum corneum, confirmed these observations. In vivo absorption of the esters was determined from the urinary excretion of total radioactivity following topical administration of 14C-labeled penetrant. Kinetic analysis of the data yielded rate constants, the ratio of which correlated acceptably with the penetrant octanol-water partition coefficient (K). The dependence of the rate constants on K was interpreted in terms of the relative affinity of the substrate for the stratum corneum compared with the viable tissue; the relationship agrees well with a previous evaluation involving structurally unrelated molecules.

In vivo percutaneous absorption of chemicals: a multiple dose study in rhesus monkeys.

The effect of daily topical application on the in vivo percutaneous absorption of benzoic acid, parathion and salicylic acid in rhesus monkeys has been investigated. The study was designed to test further the hypothesis that topical bioavailability, or body burden, of a chemical following chronic exposure may be accurately predicted from the result of a single acute-dose experiment. No significant change in percutaneous absorption from that following the initial dose was observed following the eighth daily dose of a 14-day multidose regimen for each of the three penetrants considered. The results are consistent with those of recent experiments in humans with malathion and steroids, but not entirely consistent with the results of other animal studies.