Studies in search of a suitable experimental insect model for xenodiagnosis of hosts with Chagas' disease: 2 - Attempts to upgrade the reliability and the efficacy of xenodiagnosis in chronic Chagas' disease

In order to upgrade the reliability of xenodiagnosis, attention has been directed towards population dynamics of the parasite, with particular interest for the following factors: 1. Parasite density which by itself is not a research objective, but by giving an accurate portrayal of parasite development and multiplication, has been incorporated in screening of bugs for xenodiagnosis. 2. On the assumption that food availability might increase parasite density, bugs from xenodiagnosis have been refed at biweekly intervals on chicken blood. 3. Infectivity rates and positives harbouring large parasite yields were based on gut infections, in which the parasite population comprised of all developmental forms was more abundant and easier to detect than in fecal infections, thus minimizing the probability of recording false negatives. 4. Since parasite density, low in the first 15 days of infection, increases rapidly in the following 30 days, the interval of 45 days has been adopted for routine examination of bugs from xenodiagnosis. By following the enumerated measures, all aiming to reduce false negative cases, we are getting closer to a reliable xenodiagnostic procedure. Upgrading the efficacy of xenodiagnosis is also dependent on the xenodiagnostic agent. Of 9 investigated vector species, Panstrongylus megistus deserves top priority as a xenodiagnostic agent. Its extraordinary capability to support fast development and vigorous multiplication of the few parasites, ingested from the host with chronic Chagas' disease, has been revealed by the strikingly close infectivity rates of 91.2% vs. 96.4% among bugs engorged from the same host in the chronic and acute phase of the disease respectively (Table V), the latter comporting an estimated number of 12.3 x 10[raised to the power of 3] parasites in the circulation at the time of xenodiagnosis, as reported previously by the authors (1982).

Rare genomic structural variants in complex disease: lessons from the replication of associations with obesity.

The limited ability of common variants to account for the genetic contribution to complex disease has prompted searches for rare variants of large effect, to partly explain the 'missing heritability'. Analyses of genome-wide genotyping data have identified genomic structural variants (GSVs) as a source of such rare causal variants. Recent studies have reported multiple GSV loci associated with risk of obesity. We attempted to replicate these associations by similar analysis of two familial-obesity case-control cohorts and a population cohort, and detected GSVs at 11 out of 18 loci, at frequencies similar to those previously reported. Based on their reported frequencies and effect sizes (OR≥25), we had sufficient statistical power to detect the large majority (80%) of genuine associations at these loci. However, only one obesity association was replicated. Deletion of a 220 kb region on chromosome 16p11.2 has a carrier population frequency of 2×10(-4) (95% confidence interval [9.6×10(-5)-3.1×10(-4)]); accounts overall for 0.5% [0.19%-0.82%] of severe childhood obesity cases (P = 3.8×10(-10); odds ratio = 25.0 [9.9-60.6]); and results in a mean body mass index (BMI) increase of 5.8 kg.m(-2) [1.8-10.3] in adults from the general population. We also attempted replication using BMI as a quantitative trait in our population cohort; associations with BMI at or near nominal significance were detected at two further loci near KIF2B and within FOXP2, but these did not survive correction for multiple testing. These findings emphasise several issues of importance when conducting rare GSV association, including the need for careful cohort selection and replication strategy, accurate GSV identification, and appropriate correction for multiple testing and/or control of false discovery rate. Moreover, they highlight the potential difficulty in replicating rare CNV associations across different populations. Nevertheless, we show that such studies are potentially valuable for the identification of variants making an appreciable contribution to complex disease.

Challenge of transition in the socio-professional insertion of youngsters with neurodisabilities

Patients with neurodisabilities require early management, continuing into adulthood. Thus, transition services were implemented in hospitals. To have a better support when they enter into adult life, it is useful to know the problems that they could face. The aim of this study is to evaluate their activities and to assess their insertion problems in the professional world. It is based on medical records of patients, aged 16 to 25 years, followed in the transition clinic of young adults in the Neurorehabilitation services of a tertiary centre. From 387 patients of the paediatric consultation, there are 267 patients (69%), included 224 with neurodevelopmental diseases and 43 with neuromuscular diseases. Nearly half of them (46.8%) were in a protected environment, 37.08% studied and 3.4% worked. Paradoxically, only 29.2% reported work problems. These results highlight the need to increase the integration of young adults with neuromotor disorders in the labor market.

A protocol guided by transpulmonary thermodilution and lactate levels for resuscitation of patients with severe burns.

Over-resuscitation is deleterious in many critically ill conditions, including major burns. For more than 15 years, several strategies to reduce fluid administration in burns during the initial resuscitation phase have been proposed, but no single or simple parameter has shown superiority. Fluid administration guided by invasive hemodynamic parameters usually resulted in over-resuscitation. As reported in the previous issue of Critical Care, Sánchez-Sánchez and colleagues analyzed the performance of a 'permissive hypovolemia' protocol guided by invasive hemodynamic parameters (PiCCO, Pulsion Medical Systems, Munich, Germany) and vital signs in a prospective cohort over a 3-year period. The authors' results confirm that resuscitation can be achieved with below-normal levels of preload but at the price of a fluid administration greater than predicted by the Parkland formula (2 to 4 mL/kg per% burn). The classic approach based on an adapted Parkland equation may still be the simplest until further studies identify the optimal bundle of resuscitation goals.

Nasal immunization of mice with human papillomavirus type 16 virus-like particles elicits neutralizing antibodies in mucosal secretions.

To specifically induce a mucosal antibody response to purified human papillomavirus type 16 (HPV16) virus-like particles (VLP), we immunized female BALB/c mice orally, intranasally, and/or parenterally and evaluated cholera toxin (CT) as a mucosal adjuvant. Anti-HPV16 VLP immunoglobulin G (IgG) and IgA titers in serum, saliva, and genital secretions were measured by enzyme-linked immunosorbent assay (ELISA). Systemic immunizations alone induced HPV16 VLP-specific IgG in serum and, to a lesser extent, in genital secretions but no secretory IgA. Oral immunization, even in the presence of CT, was inefficient. However, three nasal immunizations with 5 microgram of VLP given at weekly intervals to anesthetized mice induced high (>10(4)) and long-lasting (>15 weeks) titers of anti-HPV16 VLP antibodies in all samples, including IgA and IgG in saliva and genital secretions. CT enhanced the VLP-specific antibody response 10-fold in serum and to a lesser extent in saliva and genital secretions. Nasal immunization of conscious mice compared to anesthetized mice was inefficient and correlated with the absence of uptake of a marker into the lung. However, a 1-microgram VLP systemic priming followed by two 5-microgram VLP intranasal boosts in conscious mice induced both HPV16 VLP-specific IgG and IgA in secretions, although the titers were lower than in anesthetized mice given three intranasal immunizations. Antibodies in serum, saliva, and genital secretions of immunized mice were strongly neutralizing in vitro (50% neutralization with ELISA titers of 65 to 125). The mucosal and systemic/mucosal HPV16 VLP immunization protocols that induced significant titers of neutralizing IgG and secretory IgA in mucosal secretions in mice may be relevant to genital HPV VLP-based human vaccine trials.

Levels of lethal antibody during the course of infection with Schistosoma mansoni in rats and mice

Schistosoma mansoni infected hosts produce an IgG that mediates the complement-dependent killing of schistosomula in vitro. In this study, we followed the levels of serum lethal antibody during infection of rats and mice. Rats presented detectable lethal activity early in the course of infection with a peak in the 6-8th week of infection. This activity declined to non-detectable levels within 2 weeks, remaining low up to the 20-26th week. In mice, lethal antibody was not detected before 7-12 weeks of infection, but raised to higher levels, as compared to non-infected animals, up to 20-24 weeks after infection. We correlate lethal antibody and protective immunity suggesting that the antibody-mediated complement-dependent cytotoxicity to schistosomula play a role in the immunity to reinfection.

BORN AT 27 WEEKS OF GESTATION WITH CLASSICAL PKU: CHALLENGES OF DIETETIC MANAGEMENT IN A VERY PRETERM INFANT

Only few cases of classical phenylketonuria (PKU) in premature infants have been reported. Treatment of these patients is challenging due to the lack of a phenylalanine-free amino acid solution for parenteral infusion. The boy was born at 27 weeks of gestation with a weight of 1000 g (P10). He received parenteral nutrition with a protein intake of 3 g/kg/day. On day 7 he was diagnosed with classical PKU (genotype IVS10-11G>A/IVS12+ 1G>A) due to highly elevated phenylalanine (Phe) level in newborn screening (2800 micromol/L). His maximum plasma Phe level reached 3696 micromol/L. Phe intake was stopped for 4 days. During this time the boy received intravenous glucose and lipids as well as little amounts of Phe-free formula by a nasogastric tube. Due to a deficit of essential amino acids and insufficient growth, a parenteral nutrition rich in branched-chain amino-acids and relatively poor in Phe was added, in order to promote protein synthesis without overloading in Phe. Under this regimen, Phe plasma levels normalized on day 19 when intake of natural protein was started. The boy has now a corrected age of 2 years. He shows normal growth parameters and psychomotor development. Despite a long period of highly elevated Phe levels in the postnatal period our patient shows good psychomotor development. The management of premature infants with PKU depends on the child's tolerance to enteral nutrition. It demands an intensive follow-up by an experienced team and dedicated dietician. Appropriate Phe-free parenteral nutrition would be necessary especially in case of gastro-intestinal complications of prematurity.

Superior outcome of women with stage I/II cutaneous melanoma: pooled analysis of four European Organisation for Research and Treatment of Cancer phase III trials.

PURPOSE: Several studies observed a female advantage in the prognosis of cutaneous melanoma, for which behavioral factors or an underlying biologic mechanism might be responsible. Using complete and reliable follow-up data from four phase III trials of the European Organisation for Research and Treatment of Cancer (EORTC) Melanoma Group, we explored the female advantage across multiple end points and in relation to other important prognostic indicators. PATIENTS AND METHODS: Patients diagnosed with localized melanoma were included in EORTC adjuvant treatment trials 18832, 18871, 18952, and 18961 and randomly assigned during the period of 1984 to 2005. Cox proportional hazard models were used to calculate hazard ratios (HRs) and 95% CIs for women compared with men, adjusted for age, Breslow thickness, body site, ulceration, performed lymph node dissection, and treatment. RESULTS: A total of 2,672 patients with stage I/II melanoma were included. Women had a highly consistent and independent advantage in overall survival (adjusted HR, 0.70; 95% CI, 0.59 to 0.83), disease-specific survival (adjusted HR, 0.74; 95% CI, 0.62 to 0.88), time to lymph node metastasis (adjusted HR, 0.70; 95% CI, 0.51 to 0.96), and time to distant metastasis (adjusted HR, 0.69; 95% CI, 0.59 to 0.81). Subgroup analysis showed that the female advantage was consistent across all prognostic subgroups (with the possible exception of head and neck melanomas) and in pre- and postmenopausal age groups. CONCLUSION: Women have a consistent and independent relative advantage in all aspects of the progression of localized melanoma of approximately 30%, most likely caused by an underlying biologic sex difference.

Selection of patients with germline MLH1 mutated Lynch syndrome by determination of MLH1 methylation and BRAF mutation.

Lynch syndrome is one of the most common hereditary colorectal cancer (CRC) syndrome and is caused by germline mutations of MLH1, MSH2 and more rarely MSH6, PMS2, MLH3 genes. Whereas the absence of MSH2 protein is predictive of Lynch syndrome, it is not the case for the absence of MLH1 protein. The purpose of this study was to develop a sensitive and cost effective algorithm to select Lynch syndrome cases among patients with MLH1 immunohistochemical silencing. Eleven sporadic CRC and 16 Lynch syndrome cases with MLH1 protein abnormalities were selected. The BRAF c.1799T> A mutation (p.Val600Glu) was analyzed by direct sequencing after PCR amplification of exon 15. Methylation of MLH1 promoter was determined by Methylation-Sensitive Single-Strand Conformation Analysis. In patients with Lynch syndrome, there was no BRAF mutation and only one case showed MLH1 methylation (6%). In sporadic CRC, all cases were MLH1 methylated (100%) and 8 out of 11 cases carried the above BRAF mutation (73%) whereas only 3 cases were BRAF wild type (27%). We propose the following algorithm: (1) no further molecular analysis should be performed for CRC exhibiting MLH1 methylation and BRAF mutation, and these cases should be considered as sporadic CRC; (2) CRC with unmethylated MLH1 and negative for BRAF mutation should be considered as Lynch syndrome; and (3) only a small fraction of CRC with MLH1 promoter methylation but negative for BRAF mutation should be true Lynch syndrome patients. These potentially Lynch syndrome patients should be offered genetic counselling before searching for MLH1 gene mutations.

Which subgroup of patients with rheumatoid arthritis benefits from switching to rituximab versus alternative anti-tumour necrosis factor (TNF) agents after previous failure of an anti-TNF agent?

BACKGROUND: Patients with rheumatoid arthritis (RA) with an inadequate response to TNF antagonists (aTNFs) may switch to an alternative aTNF or start treatment from a different class of drugs, such as rituximab (RTX). It remains unclear in which clinical settings these therapeutic strategies offer most benefit. OBJECTIVE: To analyse the effectiveness of RTX versus alternative aTNFs on RA disease activity in different subgroups of patients. METHODS: A prospective cohort study of patients with RA who discontinued at least one aTNF and subsequently received either RTX or an alternative aTNF, nested within the Swiss RA registry (SCQM-RA) was carried out. The primary outcome, longitudinal improvement in 28-joint count Disease Activity Score (DAS28), was analysed using multivariate regression models for longitudinal data and adjusted for potential confounders. RESULTS: Of the 318 patients with RA included; 155 received RTX and 163 received an alternative aTNF. The relative benefit of RTX varied with the type of prior aTNF failure: when the motive for switching was ineffectiveness to previous aTNFs, the longitudinal improvement in DAS28 was significantly better with RTX than with an alternative aTNF (p = 0.03; at 6 months, -1.34 (95% CI -1.54 to -1.15) vs -0.93 (95% CI -1.28 to -0.59), respectively). When the motive for switching was other causes, the longitudinal improvement in DAS28 was similar for RTX and alternative aTNFs (p = 0.40). These results were not significantly modified by the number of previous aTNF failures, the type of aTNF switches, or the presence of co-treatment with a disease-modifying antirheumatic drug. CONCLUSION: This observational study suggests that in patients with RA who have stopped a previous aTNF treatment because of ineffectiveness changing to RTX is more effective than switching to an alternative aTNF.

Pharmacokinetic drug interaction potential of risperidone with cytochrome p450 isozymes as assessed by the dextromethorphan, the caffeine, and the mephenytoin test.

Two published case reports showed that addition of risperidone (1 and 2 mg/d) to a clozapine treatment resulted in a strong increase of clozapine plasma levels. As clozapine is metabolized by cytochrome P450 isozymes, a study was initiated to assess the in vivo interaction potential of risperidone on various cytochrome P450 isozymes. Eight patients were phenotyped with dextromethorphan (CYP2D6), mephenytoin (CYP2C19), and caffeine (CYP1A2) before and after the introduction of risperidone. Before risperidone, all eight patients were phenotyped as being extensive metabolizers of CYP2D6 and CYP2C19. Risperidone at dosages between 2 and 6 mg/d does not appear to significantly inhibit CYP1A2 and CYP2C19 in vivo (median plasma paraxanthine/caffeine ratios before and after risperidone: 0.65, 0.69; p = 0.89; median urinary (S)/(R) mephenytoin ratios before and after risperidone:0.11, 0.12; p = 0.75). Although dextromethorphan metabolic ratio is significantly increased by risperidone (median urinary dextromethorphan/dextrorphan ratios before and after risperidone: 0.010, 0.018; p = 0.042), risperidone can be considered a weak in vivo CYP2D6 inhibitor, as this increase is modest and none of the eight patients was changed from an extensive to a poor metabolizer. The reported increase of clozapine concentrations by risperidone can therefore not be explained by an inhibition of CYP1A2, CYP2D6, CYP2C19 or by any combination of the three.