[Fecal microbiota transplantation in recurrent Clostridium difficile infections. Framework and pharmaceutical preparation aspects]. / Le transfert de microbiote fécal lors d'infections récidivantes à Clostridium difficile. Cadre et aspects pharmacotechniques.

The fecal microbiota transplantation consists in introducing a preparation constituted by a dilution of stools of a healthy donor in the digestive tract of a patient recipient, to restore his intestinal physiological balance. This therapeutic approach was the subject of numerous studies showing its efficiency in the treatment of the recurrent infections with Clostridium difficile. The fecal microbiota transplantation has now a high level of clinical evidence, which explains that it appears in various international recommendations. In France, the fecal microbiota transplantation responds to the definition of a medication and can be executed as a pharmaceutical preparation or as an experimental drug for clinical trials under the responsibility of a hospital pharmacy. The objective of this paper is to propose a definition of a framework and to describe the methods of preparation of the fecal microbiota transplantation in the treatment of the recurrent infections with C. difficile and the interactions to consider for hospital pharmacies that do not have technical means to operate this technique.

Diversity of gut Bifidobacterium species is not altered between allergic and non-allergic French infants.

Some clinical studies have suggested a relationship between allergic diseases and gut microbiota. We aimed to study bifidobacterial colonization at species and strain levels in ten allergic French infants included at their first clinical consultation and 20 controls matching for age at sampling, mode of delivery, per partum antibiotics, type of feeding and antibiotics in the first weeks of life. The faecal microbiota was analyzed by culture methods and TTGE. Bifidobacterial species and strains were identified using multiplex PCR and Box-PCR fingerprinting. No differences were observed between groups in the number of colonized infants or in the levels of colonization by the main aerobic and anaerobic genera. All infants were colonized with high levels of Bifidobacterium except for one in each group. One to 5 Bifidobacterium species and 1 to 7 strains were observed per subject independently of allergic status and age at sampling. Our study showed the infants to be colonized by several species and strains, including several strains from the same species. This diversity in Bifidobacterium colonization was not related with the allergic status and showed that the link between Bifidobacterium colonization and allergic diseases is complex and cannot be restricted to the role attributed to Bifidobacterium species.

A 5-day course of oral antibiotics followed by faecal transplantation to eradicate carriage of multidrug-resistant Enterobacteriaceae: a randomized clinical trial.

OBJECTIVES: Intestinal carriage with extended spectrum ß-lactamase Enterobacteriaceae (ESBL-E) and carbapenemase-producing Enterobacteriaceae (CPE) can persist for months. We aimed to evaluate whether oral antibiotics followed by faecal microbiota transplantation (FMT) can eradicate intestinal carriage with ESBL-E/CPE. METHODS: Randomized, open-label, superiority trial in four tertiary-care centres (Geneva (G), Paris (P), Utrecht (U), Tel Aviv (T)). Non-immunocompromised adult patients were randomized 1: 1 to either no intervention (control) or a 5-day course of oral antibiotics (colistin sulphate 2 × 106 IU 4×/day; neomycin sulphate 500 mg 4×/day) followed by frozen FMT obtained from unrelated healthy donors. The primary outcome was detectable intestinal carriage of ESBL-E/CPE by stool culture 35-48 days after randomization (V4). ClinicalTrials.govNCT02472600. The trial was funded by the European Commission (FP7). RESULTS: Thirty-nine patients (G = 14; P = 16; U = 7; T = 2) colonized by ESBL-E (n = 36) and/or CPE (n = 11) were enrolled between February 2016 and June 2017. In the intention-to-treat analysis 9/22 (41%) patients assigned to the intervention arm were negative for ESBL-E/CPE at V4 (1/22 not receiving the intervention imputed as positive) whereas in the control arm 5/17 (29%) patients were negative (one lost to follow up imputed as negative) resulting in an OR for decolonization success of 1.7 (95% CI 0.4-6.4). Study drugs were well tolerated overall but three patients in the intervention group prematurely stopped the study antibiotics because of diarrhoea (all received FMT). CONCLUSIONS: Non-absorbable antibiotics followed by FMT slightly decreased ESBL-E/CPE carriage compared with controls; this difference was not statistically significant, potentially due to early trial termination. Further clinical investigations seem warranted.

Fecal pancreatic elastase in infants under 2 years of age.

OBJECTIVES AND METHODS: Fecal pancreatic elastase determination is of routine use in infant population presenting with a neonatal diagnosis of cystic fibrosis and in those with poor weight gain and growth, in order to precociously detect pancreatic insufficiency. However, there are few data regarding the value of one spot measure of elastase to assess pancreatic status in this population. This retrospective study reports the follow-up of fecal elastase measurement in 236 infants during the 2 first years of life. RESULTS: Fecal elastase was over 200 microg/g (i.e. normal cut-off) in a first sample in 122 patients (51.7% of patients) and below 200 microg/g in the remaining 114 patients. An alteration of elastase concentration was then observed in 18/122 infants (14.8%), leading to the diagnosis of pancreatic insufficiency at the end of the follow-up. In contrast, a normalization of fecal elastase was observed in 52 (45.6%) infants presenting with a first measurement below normal cut-off. CONCLUSION: This study shows that special attention should be given to the analysis of fecal elastase concentrations in infants as a precocious diagnosis of pancreatic insufficiency is crucial for the early introduction of a pancreatic enzyme replacement therapy which will prevent further consequence of malabsorption. One spot measure does not totally exclude pancreatic insufficiency in this population and a further control measurement of fecal elastase may be necessary.

Cancer Immunotherapy with Anti-CTLA-4 Monoclonal Antibodies Induces an Inflammatory Bowel Disease.

BACKGROUND: Therapeutic monoclonal anti-cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) antibodies are associated with immune-mediated enterocolitis. The aim of this study was to provide a detailed description of this entity. METHODS: We included patients with endoscopic signs of inflammation after anti-CTLA-4 infusions for cancer treatment. Other causes of enterocolitis were excluded. Clinical, biological and endoscopic data were recorded. A single pathologist reviewed endoscopic biopsies and colectomy specimens from 27 patients. Patients with and without enterocolitis after ipilimumab-treated melanoma were compared, to identify clinical factors associated with enterocolitis. RESULTS: Thirty-nine patients with anti-CTLA-4 enterocolitis were included (ipilimumab n = 37; tremelimumab n = 2). The most frequent symptom was diarrhoea. Ten patients had extra-intestinal manifestations. Most colonoscopies showed ulcerations involving the rectum and sigmoid, 66% of patients had extensive colitis, 55% had patchy distribution and 20% had ileal inflammation. Endoscopic colonic biopsies showed acute colitis in most patients, while half of the patients had chronic duodenitis. Thirty-five patients received steroids that led to complete clinical remission in 13 patients (37%). Twelve patients required infliximab, of whom 10 (83%) responded. Six patients underwent colectomy (perforation n = 5; toxic megacolon n = 1); one of them died postoperatively. Four patients had a persistent enterocolitis at follow-up colonoscopy. Patients with enterocolitis were more frequently prescribed NSAIDs compared with patients without enterocolitis (31 vs 5%, p = 0.003). CONCLUSIONS: Ipilimumab and tremelimumab may induce a severe and extensive form of inflammatory bowel disease. Rapid escalation to infliximab should be advocated in patients who do not respond to steroids. Patients treated with anti-CTLA-4 should be advised to avoid NSAIDs.

Fecal calprotectin and α1-antitrypsin dynamics in gastrointestinal GvHD.

In a previous study, the fecal biomarkers calprotectin and α1-antitrypsin (α1-AT) at symptom onset were reported to be significantly associated with the response to steroids in gastrointestinal GvHD (GI-GvHD). The purpose of this trial was to evaluate the dynamics of the fecal biomarkers calprotectin and α1-AT throughout the course of GvHD. Patients who were refractory to steroids had initially higher biomarker levels and in the course of GvHD demonstrated a continuous increase in fecal biomarkers. In contrast, the dynamics of calprotectin and α1-AT demonstrated low and decreasing levels in cortico-sensitive GvHD. In steroid-refractory patients who received a second line of treatment, the biomarker levels at the beginning of second-line treatment did not predict the subsequent response. Nevertheless, calprotectin levels progressively decreased in subsequent responders, whereas non-responders demonstrated continuously high levels of calprotectin. α1-AT values correlated to a lesser extent with the response to second-line treatment and remained elevated in both non-responders and responders. In conclusion, calprotectin monitoring can be of use in the management of immunosuppressive treatment in GI-GvHD.

Determination of anti-Cryptosporidium coproantibodies by time-resolved immunofluorometric assay.

The role of the mucosal immune response against Cryptosporidium has been suggested by studies on the therapeutic effects of hyperimmune colostrum. In order to study the intestinal response to this infection, we have developed a sandwich-type time-resolved immunofluorometric assay for the determination of anti-Cryptosporidium coproantibodies. This assay has the inherent sensitivity of an immunoassay without the problems due to background responses from other biological compounds, and is thus suitable for faecal samples. The intra-assay coefficients of variation (5.1%, 4.6%, and 5.8% for immunoglobulins (Ig) A, M and G respectively), inter-assay coefficients of variation (9.4%; 10.5% and 12.2% for IgA, IgM and IgG, respectively) and specificity (100% for all 3 isotypes) were all satisfactory. Using this assay to study 12 patients with the acquired immune deficiency syndrome (AIDS) who were infected with cryptosporidiosis, we found a marked elevation of anti-Cryptosporidium IgA and IgM coproantibody titres relative to 18 healthy control values, but no correlation with the gravity of the infection in terms of oocyst shedding. These results suggest that a non-protective mucosal immune response develops to Cryptosporidium in AIDS patients.

Characterisation of the main molecular forms of human fecal immunoglobulin A.

The molecular composition of fecal IgA is poorly documented, although it is of theoretical and practical importance to determine the different forms of IgA in faeces. Two main molecular forms were isolated by successive steps of ion-exchange chromatography and gel-filtration. The first consisted of secretory IgA dimers dissociating into slightly lower molecular mass forms under the influence of the electric field during electrophoresis. The other contained cleaved-IgA complexed with alpha 1-antitrypsin, that is considered to be a serum origin marker. These results confirm that secretory IgA are relatively resistant to digestive enzymes in vivo, and suggest that alpha 1-antitrypsin-bound fragments originate from serum IgA monomers. Analysis of the proportions of these forms may be of value in the investigation of gut diseases.

High faecal calprotectin concentrations in newborn infants.

BACKGROUND: Calprotectin, a major component of soluble cytosolic proteins in human neutrophil granulocytes, is excreted in excess in stools during inflammatory bowel disease in adults and children. Faecal calprotectin concentrations are also higher during the first year of life than in adults. OBJECTIVES: To measure faecal calprotectin concentrations in the neonatal period and define reference values according to the mode of feeding: standard infant formula, prebiotic infant formula (Calisma, Blédina SA, France), or breast feeding. PATIENTS AND METHODS: A prospective study was carried out over three months in 69 full term, healthy newborns with a median gestational age of 39.8 weeks (range 37-41.5) and a birth weight of 3300 g (range 2600-4460). Three groups were formed depending on the mode of feeding: group 1 (n = 18) received a standard infant formula, group 2 (n = 19) the prebiotic infant formula, and group 3 (n = 32) was breast fed. One stool sample was taken from each newborn on day 4 (3-7), and faecal calprotectin analysed using a commercial enzyme linked immunoassay (Calprest, Eurospital, Italy). RESULTS: Faecal calprotectin concentrations (median 167 micro g/g) were higher than reference values in healthy adults. The concentration was below the upper reference limit for adults (50 micro g/g) for three infants only, one fed the standard formula and two fed the prebiotic formula. Concentrations did not differ significantly according to method of feeding. CONCLUSIONS: Compared with healthy adults, newborns have high calprotectin concentrations in the first days of life. There is no obvious influence of the mode of feeding.

Low levels of pancreatic elastase 1 in stools of preterm infants.

The amount of faecal pancreatic enzyme elastase 1 was significantly lower in 42 preterm newborns than in 12 full term babies at day 2 (89 (3-539) v 354 (52-600) microg/g, p<0.0007) and day 5 (164 (3-600) v 600 (158-600) microg/g, p<0.05) and correlated positively with total nutrient intake during the first week of life in preterm infants. This should probably be taken into account during early feeding.

[Detection of protein p53 in the stool of patients with colorectal cancer]. / Détection de la protéine p53 dans les selles de malades atteints de cancer colo-rectal.

BACKGROUND AND AIMS: Mutations of TP53, a tumor suppressor gene, are found in 60% to 70% of colorectal cancers. These mutations usually induce an overexpression caused by modification of the p53 protein conformation. The aim of this study was to investigate whether stool specimens of patients with colorectal cancer contain increased amounts of p53 protein. METHODS: p53 protein was measured using a sandwich enzyme immunoassay in the stool specimens of 52 patients: 25 with colorectal cancer, 4 with colorectal adenomas and 23 apparently free of gastrointestinal disease. Results were expressed as pg/mg of total protein. The presence of fecal occult-blood was searched using Hemoccult II and Hemolex (an immunochemical assay for human hemoglobin). RESULTS: Median concentrations of stool p53 protein were 16.6 pg/mg (range: 0-591 pg/mg) in patients with colorectal cancers, 39.1 pg/mg (range: 5-72 pg/mg) in patients with adenomas and 5.9 pg/mg (range: 0-65 pg/mg) in control subjects. Resection of colorectal cancers caused a marked decrease of stool p53 protein concentrations. When the cut-off value for stool p53 protein was set at 60 pg/mg of fecal protein (concentrations over the 95th percentile), the positivity of the assay was independent of tumor size and Astler-Coller stage, but weakly associated with rectal location of cancer. The sensitivity of stool p53 protein for colorectal cancer was 44%, and the specificity was 96%. In contrast, the sensitivity of Hemoccult II and Hemolex tests was 48% and 44%, whereas their specificity was 91% and 96%, respectively. CONCLUSION: The detection of p53 protein is achievable in stool, but this assay is not more efficient than fecal occult blood tests for detection of colorectal cancer.

A high-yield outbred suckling mouse model of cryptosporidiosis.

Outbred suckling mice (NMRI strain) were used as hosts. They were initially inoculated with oocysts of human origin, and subsequently with parasites recovered from the mouse ileal mucosa. Cryptosporidia were counted in an aliquot of whole-ileum homogenate. Parasite load was expressed as cryptosporidia per centimeter of ileum. Serial passage of C. parvum in NMRI mouse litters led to a gradual amplification of parasite burden relative to animals initially inoculated with the human isolate.

[Faecal occult blood testing: comparison of a latex agglutination test (Hemolex) and an immunoturbidimetric test (QuikRead FOB)]. / Recherche de sang occulte dans les selles: analyse comparative d'un test d'agglutination de particules de latex sensibilisées (Hémolex) et d'un test immunoturbidimétrique (QuikRead FOB).

UNLABELLED: Occult blood detection is the most prescribed faecal examination. AIM: To compare results obtained with the latex agglutination test Hémolex LA (Orion diagnostica, Finlande) with those given by an immuno-turbidimetric test which allows an automatic reading (QuikRead FOB, Orion diagnostica, Finlande). MATERIAL AND METHODS: this prospective study was carried out in 140 patients. The reference method was the latex agglutination test, Hemolex LA performed on stool extract obtained through weighting samples. On the base of the results, samples were separated into 2 groups: positive (n = 45) and negative (n = 95). As the QuikRead FOB test indicated a stool extract obtained through a sampling set, such an extraction was performed before Hemolex LA et QuikRead FOB testing. RESULTS: all the 95 samples from the negative group gave similar results with the 3 methods. In contrast, 12/45 of the positive samples gave conflicting results, 11 results were negative with the 2 tests performed on stool extract obtained via sampling set, 1 result was negative with the QuikRead FOB method only. DISCUSSION: analytical performance were similar with the 2 methods and discrepancies observed wi-thin the positive group were mainly related to the extraction method.

[Time-resolved fluorometry: principles and applications in clinical biology]. / Fluorimétrie en temps retardé: principe et applications en biologie clinique.

Time-resolved fluorometric assay is based on lanthanide fluorescence. This time-resolved fluorescence has a narrow-band emission line whose wavelength differs from that of emission-pulsed light and has a long decay-time. These characteristics make it possible to avoid background interference from sample constituents (protein, light-scattering particles, etc). Europium and its chelates are the most commonly used lanthanides. The europium-labelling of antigens or antibodies is followed by immunoassay. In the final step, fluorescence is measured, after enhancement, as counts per second. This assay has several advantages, including a wide working range, high sensitivity and good practicability. The method has widespread applications in the field of immunoassays in both clinical and research laboratories. The use of non-radioactive europium-labelled probes and the development of simultaneous multiple tests are possible future orientations.

Detection of occult blood in stools: comparison of three gaiac tests and a latex agglutination test.

The detection of occult blood in the stools is the only simple screening method for colorectal cancer. The aim of this study was to compare the results obtained with the new Hemolex kit (Orion diagnostica, Fumouze France) with those given by three gaiac tests--Hemoccult (Smithkline diagnostics), Hemofec (Boehringer Mannheim) and Hemopreuve (Fumouze) of 165 stools from patients without special diet. Seventy-one patients with at least two positive gaiac tests or a positive Hemolex test underwent colonoscopy followed, if negative, by fibroscopy: 28 had lesions of the lower digestive tract and five of the upper digestive tract. Sensitivity, specificity and negative and positive predictive value were of 70, 98, 91 and 92% respectively for Hemolex; 82, 74, 94 and 44% for Hemoccult; 94, 67, 98 and 42% for Hemopreuve and 91, 73, 97 and 46% for Hemofec. The results obtained in this study confirm the value of the Hemolex test for the detection of human occult blood in the stools whereas the gaiac tests used are influenced by dietary components (unless restricted), explaining their poor positive predictive value. In conclusion, due to their good negative predictive values, the authors recommend that screening for colorectal tumours should be based on the use of two or three gaiac tests which should be confirmed, when positive, by an immunological test for human hemoglobin.

[Element of digestive pathophysiology and fecal analysis]. / Elément de physiopathologie digestive et coprologie fonctionnelle.

Fecal analysis includes qualitative and quantitative studies which allows quantification and labelling of numerous pathophysiologic phenomenona. Malabsorption and over-absorption of water and electrolytes give rise to six types of watery diarrheas, and two types of constipations; malabsorption of nutriments and maldigestion of food, give rise to two types of fatty and nitrogenous diarrheas with metabolic consequences. Fecal analysis often discriminates organic from non-organic diseases and brings informations on increase or decrease of caloric losses, to the nutritionist. Microscopic observations which requires a high degree of competence and experience, allows the recognition of malabsorption/maldigestion phenomenona, of fortuitous presence of parasites and a good interpretation of a fecal file.

[New fecal markers: recent developments and perspectives]. / Les nouveaux marqueurs disponibles en coprologie fonctionnelle et perspectives.

Fecal occult blood testing is the most widely prescribed screening test for colorectal cancer. Recent development of immunological tests has increased specificity. Fecal DNA analysis opens up a new field for early detection of this widespread neoplasia. Inflammatory bowel disease is another important area where the development of fecal markers provides an interesting alternative to the gold standard but costly and invasive endoscopic investigations with histological analysis of biopsy specimens. Fecal TNFalpha and calprotectin can now be proposed to distinguish organic from non-organic intestinal disease, so select candidates for further investigations, and to assess disease activity. Measurement of fecal elastase provides real progress in screening for exocrine pancreatic insufficiency in patients with malabsorption syndrome. The development of non-invasive fecal markers is thus of increasing interest, providing data about the entire gastrointestinal tract useful for screening and individual patient management.

[Quality control in coprology]. / L'assurance de qualité en coprologie fonctionnelle.

Quality control in medical laboratories was defined in guidelines for good execution of laboratory analyses issued by the French health authorities in 1994. Application of these guidelines is difficult in coprology because the sample is a complex heterogeneous matrix which varies with disease, surgery, food intake, and treatment. In addition, commercial quality control kits are not available for stool biochemical analyses and a national quality control program has not been established. We thus developed our own fecal quality control technique using pooling lyophylized stool samples. Manual or partially automated methods are used in coprology, leading to a long pre-analysis phase which is not always taken into account in quality control. This implies the need for complementary tools to insure the quality of coprology analyses. For example, semi-quantitative microscopic lipid analysis can be used as an internal standard for a given specimen. Quality assurance also involves a post-analytical phase where results obtained for a given specimen are compared with other available data and interpreted in light of the patient's clinical and therapeutic status. This quality assurance strategy enables accurate reliable results useful for long-term patient management.

[Cryptosporidium parvum: functional study of the intestinal malabsorption syndrome]. / Cryptosporidium parvum: étude fonctionnelle du syndrome de malabsorption intestinale.

Cryptosporidiosis is an important cause of diarrhea associated with growth retardation in children and severe malnutrition in immunocompromised patients. The pathophysiology is poorly understood. In the suckling rat model, we show that C. parvum infection impairs net electrogenic transport across the ileal mucosa without involvement of prostaglandins, as well as trans- and paracellular permeability and leucine and glutamate absorption. These results provide evidence for the development of an intestinal malabsorptive syndrome during cryptosporidiosis. Unspecific process such as villous atrophy and inflammatory cytokines secretion should be regarded as possible mediators of this syndrome. However, specific mechanisms have to be considered since C. parvum induces a rearrangement of the host enterocyte cytoskeleton which might impaired intracellular trafficking thus reducing the membrane expression of nutrient transporters. Infection and malnutrition are known to be tightly associated, making each other worse. As no specific efficient therapy exists, cryptosporidiosis-induced malnutrition must be taken into account when establishing therapeutic scheme.