Raman and infra-red microspectroscopy: towards quantitative evaluation for clinical research by ratiometric analysis

Biomolecular structure elucidation is one of the major techniques for studying the basic processes of life. These processes get modulated, hindered or altered due to various causes like diseases, which is why biomolecular analysis and imaging play an important role in diagnosis, treatment prognosis and monitoring. Vibrational spectroscopy (IR and Raman), which is a molecular bond specific technique, can assist the researcher in chemical structure interpretation. Based on the combination with microscopy, vibrational microspectroscopy is currently emerging as an important tool for biomedical research, with a spatial resolution at the cellular and sub-cellular level. These techniques offer various advantages, enabling label-free, biomolecular fingerprinting in the native state. However, the complexity involved in deciphering the required information from a spectrum hampered their entry into the clinic. Today with the advent of automated algorithms, vibrational microspectroscopy excels in the field of spectropathology. However, researchers should be aware of how quantification based on absolute band intensities may be affected by instrumental parameters, sample thickness, water content, substrate backgrounds and other possible artefacts. In this review these practical issues and their effects on the quantification of biomolecules will be discussed in detail. In many cases ratiometric analysis can help to circumvent these problems and enable the quantitative study of biological samples, including ratiometric imaging in 1D, 2D and 3D. We provide an extensive overview from the recent scientific literature on IR and Raman band ratios used for studying biological systems and for disease diagnosis and treatment prognosis.

High resolution cortical bone thickness measurement from clinical CT data.

The distribution of cortical bone in the proximal femur is believed to be a critical component in determining fracture resistance. Current CT technology is limited in its ability to measure cortical thickness, especially in the sub-millimetre range which lies within the point spread function of today's clinical scanners. In this paper, we present a novel technique that is capable of producing unbiased thickness estimates down to 0.3mm. The technique relies on a mathematical model of the anatomy and the imaging system, which is fitted to the data at a large number of sites around the proximal femur, producing around 17,000 independent thickness estimates per specimen. In a series of experiments on 16 cadaveric femurs, estimation errors were measured as -0.01+/-0.58mm (mean+/-1std.dev.) for cortical thicknesses in the range 0.3-4mm. This compares with 0.25+/-0.69mm for simple thresholding and 0.90+/-0.92mm for a variant of the 50% relative threshold method. In the clinically relevant sub-millimetre range, thresholding increasingly fails to detect the cortex at all, whereas the new technique continues to perform well. The many cortical thickness estimates can be displayed as a colour map painted onto the femoral surface. Computation of the surfaces and colour maps is largely automatic, requiring around 15min on a modest laptop computer.

The descriptive epidemiology of congenital and acquired cryptorchidism in a UK infant cohort.

INTRODUCTION: Recent studies in other European countries suggest that the prevalence of congenital cryptorchidism continues to increase. This study aimed to explore the prevalence and natural history of congenital cryptorchidism in a UK centre. METHODS: Between October 2001 and July 2008, 784 male infants were born in the prospective Cambridge Baby Growth Study. 742 infants were examined by trained research nurses at birth; testicular position was assessed using standard techniques. Follow-up assessments were completed at ages 3, 12, 18 and 24 months in 615, 462, 393 and 326 infants, respectively. RESULTS: The prevalence of cryptorchidism at birth was 5.9% (95% CI 4.4% to 7.9%). Congenital cryptorchidism was associated with earlier gestational age (p<0.001), lower birth weight (p<0.001), birth length (p<0.001) and shorter penile length at birth (p<0.0001) compared with other infants, but normal size after age 3 months. The prevalence of cryptorchidism declined to 2.4% at 3 months, but unexpectedly rose again to 6.7% at 12 months as a result of new cases. The cumulative incidence of "acquired cryptorchidism" by age 24 months was 7.0% and these cases had shorter penile length during infancy than other infants (p = 0.003). CONCLUSIONS: The prevalence of congenital cryptorchidism was higher than earlier estimates in UK populations. Furthermore, this study for the first time describes acquired cryptorchidism or "ascending testis" as a common entity in male infants, which is possibly associated with reduced early postnatal androgen activity.

Transposon mutagenesis in a hyper-invasive clinical isolate of Campylobacter jejuni reveals a number of genes with potential roles in invasion.

Transposon mutagenesis has been applied to a hyper-invasive clinical isolate of Campylobacter jejuni, 01/51. A random transposon mutant library was screened in an in vitro assay of invasion and 26 mutants with a significant reduction in invasion were identified. Given that the invasion potential of C. jejuni is relatively poor compared to other enteric pathogens, the use of a hyper-invasive strain was advantageous as it greatly facilitated the identification of mutants with reduced invasion. The location of the transposon insertion in 23 of these mutants has been determined; all but three of the insertions are in genes also present in the genome-sequenced strain NCTC 11168. Eight of the mutants contain transposon insertions in one region of the genome (approximately 14 kb), which when compared with the genome of NCTC 11168 overlaps with one of the previously reported plasticity regions and is likely to be involved in genomic variation between strains. Further characterization of one of the mutants within this region has identified a gene that might be involved in adhesion to host cells.

Randomized, double-blind, placebo-controled clinical trial of sublingual immunotherapy in natural rubber latex allergic patients

Trial registration number: CTRN12611000543987

Human Papillomavirus (HPV) genotype 18 variants in patients with clinical manifestations of HPV related infections in Bilbao, Spain

Background:Human papillomavirus (HPV) variants differ in their biological and chemical properties, and therefore, may present differences in pathogenicity. Most authors classified variants based on the phylogenetic analysis of L1 region. Nevertheless, recombination in HPV samples is becoming a usual finding and thus, characterizing genetic variability in other regions should be essential. Objectives:We aimed to characterize the genetic variability of HPV 18 in 5 genomic regions: E6, E7, E4, L1 and the Upstream Regulatory Region (URR), working with both single infection and multiple HPV infection samples. Furthermore, we aimed to assess the prevalence of HPV 18 variants in our region and look for possible existence of recombination as well as analyze the relationship between these variants and the type of lesion. Methods: From 2007 to 2010, Clinical Microbiology and Infection Control Department analyzed 44 samples which were positive for HPV 18. Genetic variability was determined in PCR products and variants were assigned to European, Asian-amerindian or African lineage. Recombination and association of variants with different types of lesion was studied. Results: Genetic analysis of the regions revealed a total of 56 nucleotide variations. European, African and Asian-amerindian variants were found in 25/44 (56.8%), 10/44 (22.7%) and 5/44 (11.4%) samples, respectively. We detected the presence of recombinant variants in 2/44 (4.5%) cases. Samples taken from high-grade squamous intraepithelial lesions (H-SIL) only presented variants with specific-african substitutions. Conclusions: Multiple HPV infection, non-european HPV variants prevalence and existence of recombination are considered risk factors for HPV persistence and progression of intraepithelial abnormalities, and therefore, should be taken into consideration in order to help to design and optimize diagnostics protocols as well as improve epidemiologic studies. Our study is one of the few studies in Spain which analyses the genetic variability of HPV18 and we showed the importance of characterizing more than one genomic region in order to detect recombination and classify HPV variants properly

Use of generalised additive models to categorise continuous variables in clinical prediction

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Malaria Vaccine Adjuvants: Latest Update and Challenges in Preclinical and Clinical Research

There is no malaria vaccine currently available, and the most advanced candidate has recently reported a modest 30% efficacy against clinical malaria. Although many efforts have been dedicated to achieve this goal, the research was mainly directed to identify antigenic targets. Nevertheless, the latest progresses on understanding how immune system works and the data recovered from vaccination studies have conferred to the vaccine formulation its deserved relevance. Additionally to the antigen nature, the manner in which it is presented (delivery adjuvants) as well as the immunostimulatory effect of the formulation components (immunostimulants) modulates the immune response elicited. Protective immunity against malaria requires the induction of humoral, antibody-dependent cellular inhibition (ADCI) and effector and memory cell responses. This review summarizes the status of adjuvants that have been or are being employed in the malaria vaccine development, focusing on the pharmaceutical and immunological aspects, as well as on their immunization outcomings at clinical and preclinical stages.

Clinical factors associated with a Candida albicans Germ Tube Antibody positive test in Intensive Care Unit patients

Background: Poor outcomes of invasive candidiasis (IC) are associated with the difficulty in establishing the microbiological diagnosis at an early stage. New scores and laboratory tests have been developed in order to make an early therapeutic intervention in an attempt to reduce the high mortality associated with invasive fungal infections. Candida albicans IFA IgG has been recently commercialized for germ tube antibody detection (CAGTA). This test provides a rapid and simple diagnosis of IC (84.4% sensitivity and 94.7% specificity). The aim of this study is to identify the patients who could be benefited by the use of CAGTA test in critical care setting. Methods: A prospective, cohort, observational multicentre study was carried out in six medical/surgical Intensive care units (ICU) of tertiary-care Spanish hospitals. Candida albicans Germ Tube Antibody test was performed twice a week if predetermined risk factors were present, and serologically demonstrated candidiasis was considered if the testing serum dilution was >= 1: 160 in at least one sample and no other microbiological evidence of invasive candidiasis was found. Results: Fifty-three critically ill non-neutropenic patients (37.7% post surgery) were included. Twenty-two patients (41.5%) had CAGTA-positive results, none of them with positive blood culture for Candida. Neither corrected colonization index nor antifungal treatment had influence on CAGTA results. This finding could corroborate that the CAGTA may be an important biomarker to distinguish between colonization and infection in these patients. The presence of acute renal failure at the beginning of the study was more frequent in CAGTA-negative patients. Previous surgery was statistically more frequent in CAGTA-positive patients. Conclusions: This study identified previous surgery as the principal clinical factor associated with CAGTA-positive results and emphasises the utility of this promising technique, which was not influenced by high Candida colonization or antifungal treatment. Our results suggest that detection of CAGTA may be important for the diagnosis of invasive candidiasis in surgical patients admitted in ICU.

Randomised, crossover clinical trial, in healthy volunteers, to compare the systemic availability of two topical intranasal budesonide formulations

Background: Budesonide has a long history as intranasal drug, with many marketed products. Efforts should be made to demonstrate the therapeutic equivalence and safety comparability between them. Given that systemic availability significantly varies from formulations, the clinical comparability of diverse products comes to be of clinical interest and a regulatory requirement. The aim of the present study was to compare the systemic availability, pharmacodynamic effect, and safety of two intranasal budesonide formulations for the treatment of rhinitis. Methods: Eighteen healthy volunteers participated in this randomised, controlled, crossover, clinical trial. On two separated days, subjects received a single dose of 512 mu g budesonide (4 puffs per nostril) from each of the assayed devices (Budesonida nasal 64 (R), Aldo-Union, Spain and Rhinocort 64 (R), AstraZeneca, Spain). Budesonide availability was determined by the measurement of budesonide plasma concentration. The pharmacodynamic effect on the hypothalamic-adrenal axis was evaluated as both plasma and urine cortisol levels. Adverse events were tabulated and described. Budesonide availability between formulations was compared by the calculation of 90% CI intervals of the ratios of the main pharmacokinetic parameters describing budesonide bioavailability. Plasma cortisol concentration-time curves were compared by means of a GLM for Repeated Measures. Urine cortisol excretion between formulations was compared through the Wilcoxon's test. Results: All the enroled volunteers successfully completed the study. Pharmacokinetic parameters were comparable in terms of AUC(t) (2.6 +/- 1.5 vs 2.2 +/- 0.7), AUCi (2.9 +/- 1.5 vs 2.4 +/- 0.7), t(max) (0.4 +/- 0.1 vs 0.4 +/- 0.2), C(max)/AUC(i) (0.3 +/- 0.1 vs 0.3 +/- 0.0), and MRT (5.0 +/- 1.4 vs 4.5 +/- 0.6), but not in the case of C(max) (0.9 +/- 0.3 vs 0.7 +/- 0.2) and t(1/2) (3.7 +/- 1.8 vs 2.9 +/- 0.4). The pharmacodynamic effects, measured as the effect over plasma and urine cortisol, were also comparables between both formulations. No severe adverse events were reported and tolerance was comparable between formulations. Conclusion: The systemic availability of intranasal budesonide was comparable for both formulations in terms of most pharmacokinetic parameters. The pharmacodynamic effect on hypothalamic-pituitary-adrenal axis was also similar. Side effects were scarce and equivalent between the two products. This methodology to compare different budesonide-containing devices is reliable and easy to perform, and should be recommended for similar products intented to be marketed or already on the market.