Comparison of methods available for identification of Mycobacterium chimaera.

OBJECTIVES: Mycobacterium chimaera is a recently described nontuberculous mycobacterium belonging to the Mycobacterium avium complex (MAC). Because this species is implicated in a worldwide outbreak due to contaminated heater-cooler unit water tanks during open-heart surgery, it has become mandatory for clinical microbiology laboratories to be able to differentiate M. chimaera from the other MAC species, especially M. intracellulare. Such identification has so far been restricted to specialized laboratories because it required the analysis of several gene sequences. The aim of this study was to evaluate commercial methods for identifying M. chimaera with regard to the reference gene sequencing ITS, the internal transcribed spacer 16-23S. METHODS: Forty-seven clinical and environmental isolates including 41 MAC were identified by (a) PCR sequencing of the ITS and hsp65 genes, (b) three molecular biology kits (INNO-LiPA Mycobacteria, GenoType Mycobacterium CM and GenoType NTM-DR) and (c) matrix-assisted desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using Microflex LT. RESULTS: There was a high concordance for species determination between the reference ITS sequencing and the GenoType NTM-DR test (39/41, 95%), the INNO-LiPA Mycobacteria test (38/41, 93%) and the hsp65 sequencing (38/41, 93%). The GenoType Mycobacterium CM test did not distinguish M. chimaera from M. intracellulare. MALDI-TOF MS distinguished two M. chimaera-M. intracellulare groups separated from M. avium and from the other mycobacterial species on a score-oriented dendrogram, but it also failed to differentiate the two species. CONCLUSIONS: INNO-LiPA Mycobacteria and GenoType NTM-DR are efficient assays for M. chimaera identification in clinical microbiology laboratories.

Long-Term Outcomes of Intestinal and Multivisceral Transplantation at a Single Center in Argentina.

BACKGROUND: Intestinal failure (IF) patients received parenteral nutrition (PN) as the only available therapy until intestinal transplantation (ITx) evolved as an accepted treatment. The aim of this article is to report the long-term outcomes of a series of ITx performed in pediatric and adult patients at a single center 9 years after its creation. PATIENTS AND METHODS: This is a retrospective analysis of the ITx performed between May 2006 and January 2015. Diagnoses, pre-ITx mean time on PN, indications for ITx, time on the waiting list for types of ITx, mean total ischemia time, and warm ischemia time, time until PN discontinuation, incidence of acute and chronic rejection, and 5-year actuarial patient survival are reported. RESULTS: A total of 42 patients received ITx; 80% had short gut syndrome (SG); the mean time on PN was 1620 days. The main indication for ITx was lack of central venous access followed by intestinal failure-associated liver disease (IFALD) and catheter-related infectious complications. The mean time on the waiting list was 188 days (standard deviation, ±183 days). ITx were performed in 26 children and 14 adults. In all, 32 procedures were isolated ITx (IITX); 10 were multiorgan Tx (MOT; 3 combined, 7 multivisceral Tx (MVTx), 1 modified MVTx and 2 with kidney); 2 (4.7 %) were retransplantations: 1 IITx, 1 MVTx, and 5 including the right colon. Thirteen patients (31%) received abdominal rectus fascia. All procedures were performed by the same surgical team. Total ischemia time was 7:53 ± 2:04 hours, and warm ischemia time was 40.2 ± 10.5 minutes. The mean length of implanted intestine was 325 ± 63 cm. Bishop-Koop ileostomy was performed in 67% of cases. In all, 16 of 42 Tx required early reoperations. The overall mean follow-up time was 41 ± 35.6 months. The mean time to PN discontinuation after Tx was 68 days (P = .001). The total number of acute cellular rejection (ACR) episodes until the last follow-up was 83; the total number of grafts lost due to ACR was 4; and the total graft lost due to chronic rejection was 3. At the time of writing, the overall 5-year patient survival is 55% (65% for IITx vs 22% for MOT; P = .0001); 60% for pediatric recipients vs 47% for adults (P = NS); 64% when the indication for ITx was SG vs 25% for non-SG (P = .002). CONCLUSIONS: At this center, candidates with SG, in the absence of IFALD requiring IITx, showed the best long-term outcomes, independent of recipient age. A multidisciplinary approach is mandatory for the care of intestinal failure patients, to sustain a rehabilitation and transplantation program over time.

Case Report: Multivisceral Transplantation for an Extensive Cystic Lymphangioma of the Mesenteric Root.

BACKGROUND: We report the case of a 7-year-old girl with intestinal failure owing to a cystic lymphangioma compromising the root of the mesentery, not amenable to resection, leading to intestinal failure. Oncologic treatment was attempted to reduce tumor size with no response; therefore, she was listed for multivisceral transplantation. PROCEDURE: Resection of the tumor required resection of all abdominal organs with vascular inflow and outflow. A multivisceral graft (liver, stomach, duodenum-pancreas and spleen complex, small bowel, and right colon) was implanted. For vascular reconstruction, donor's superior vena cava was sutured to the recipient's suprahepatic veins in a common patch. For arterial inflow, an arterial conduit was placed directly to the infrarenal aorta, and sutured to an aortic patch of the graft. Cold ischemia time was 8:45 hours; warm ischemia time was 35 minutes. A double-layer gastrogastric anastomosis and piloroplasty was made; and the distal reconstruction was performed with ileocolic side-to-end anastomosis that allowed to perform of a Bishop-Koop ileostomy for endoscopic monitoring. OUTCOME: The patient recovered well after the procedure and was discharged 36 days after transplantation with intestinal sufficiency. To the best of our knowledge, this is the first report describing cystic lymphangioma as an indication for multivisceral transplantation.

Case Report: Spleen-preserving Multivisceral Transplant for Peutz-Jeghers Syndrome.

CASE REPORT: A 24-year-old man diagnosed with Peutz-Jeghers syndrome as a child underwent multiple surgeries owing to intussusception. Pretransplant workup showed >150 polyps along the gastrointestinal (GI) tract, some of them with high-grade dysplasia. Despite having intestinal sufficiency, a modified multivisceral transplantation was offered. PROCEDURE: An 18-year-old donor was procured using University of Wisconsin solution. The recipient's surgery started with a midline incision. Mobilization of the right colon and the root of the mesentery was done to isolate the superior mesenteric artery. The same maneuver was done with the left and sigmoid colon. The common bile duct was then isolated and transected at the cystic duct level. The abdominal portion of the esophagus and the proximal stomach were isolated and divided at the gastroesophageal junction. After that, the pancreas was mobilized, preserving the spleen with the splenic vessels. The distal GI tract was transacted at the level of the proximal rectum. For engraftment, an arterial conduit was placed in the infrarenal aorta and anastomosed to the graft's aortic patch. End-to-side portal reconstruction was made at the level of the portal vein, allowing performing a duct-to-duct biliary reconstruction over a 5-Fr T-tube. A hand-sewn gastrogastric anastomosis and piloroplasty were performed; the distal anastomosis was done with circular staplers. A gastrojejunostomy and a loop ileostomy were the final steps of the procedure. RESULTS: The patient stayed in intensive care for 2 days and enteral feeds were started on day 7. Currently, 23 months after transplant he is alive with an excellent quality of life.

Viral persistence in surface and drinking water: Suitability of PCR pre-treatment with intercalating dyes.

After many outbreaks of enteric virus associated with consumption of drinking water, the study of enteric viruses in water has increased significantly in recent years. In order to better understand the dynamics of enteric viruses in environmental water and the associated viral risk, it is necessary to estimate viral persistence in different conditions. In this study, two representative models of human enteric viruses, adenovirus 41 (AdV 41) and coxsackievirus B2 (CV-B2), were used to evaluate the persistence of enteric viruses in environmental water. The persistence of infectious particles, encapsidated genomes and free nucleic acids of AdV 41 and CV-B2 was evaluated in drinking water and surface water at different temperatures (4 °C, 20 °C and 37 °C). The infectivity of AdV 41 and CV-B2 persisted for at least 25 days, whatever the water temperature, and for more than 70 days at 4 °C and 20 °C, in both drinking and surface water. Encapsidated genomes persisted beyond 70 days, whatever the water temperature. Free nucleic acids (i.e. without capsid) also were able to persist for at least 16 days in drinking and surface water. The usefulness of a detection method based on an intercalating dye pre-treatment, which specifically targets preserved particles, was investigated for the discrimination of free and encapsidated genomes and it was compared to virus infectivity. Further, the resistance of AdV 41 and CV-B2 against two major disinfection treatments applied in drinking water plants (UV and chlorination) was evaluated. Even after the application of UV rays and chlorine at high doses (400 mJ/cm(2) and 10 mg.min/L, respectively), viral genomes were still detected with molecular biology methods. Although the intercalating dye pre-treatment had little use for the detection of the effects of UV treatment, it was useful in the case of treatment by chlorination and less than 1 log10 difference in the results was found as compared to the infectivity measurements. Finally, for the first time, the suitability of intercalating dye pre-treatment for the estimation of the quality of the water produced by treatment plants was demonstrated using samples from four drinking-water plants and two rivers. Although 55% (27/49) of drinking water samples were positive for enteric viruses using molecular detection, none of the samples were positive when the intercalating dye pre-treatment method was used. This could indicate that the viruses that were detected are not infectious.

Large scale survey of enteric viruses in river and waste water underlines the health status of the local population.

Although enteric viruses constitute a major cause of acute waterborne diseases worldwide, environmental data about occurrence and viral load of enteric viruses in water are not often available. In this study, enteric viruses (i.e., adenovirus, aichivirus, astrovirus, cosavirus, enterovirus, hepatitis A and E viruses, norovirus of genogroups I and II, rotavirus A and salivirus) were monitored in the Seine River and the origin of contamination was untangled. A total of 275 water samples were collected, twice a month for one year, from the river Seine, its tributaries and the major WWTP effluents in the Paris agglomeration. All water samples were negative for hepatitis A and E viruses. AdV, NVGI, NVGII and RV-A were the most prevalent and abundant populations in all water samples. The viral load and the detection frequency increased significantly between the samples collected the most upstream and the most downstream of the Paris urban area. The calculated viral fluxes demonstrated clearly the measurable impact of WWTP effluents on the viral contamination of the Seine River. The viral load was seasonal for almost all enteric viruses, in accordance with the gastroenteritis recordings provided by the French medical authorities. These results implied the existence of a close relationship between the health status of inhabitants and the viral contamination of WWTP effluents and consequently surface water contamination. Subsequently, the regular analysis of wastewater could serve as a proxy for the monitoring of the human viruses circulating in both a population and surface water.

Phylogenetic analyses of symbiotic genes and characterization of functional traits of Mesorhizobium spp. strains associated with the promiscuous species Acacia seyal Del.

AIMS: To assess the phenotypic, symbiotic and genotypic diversity scope of Mesorhizobium spp. strains associated with Acacia seyal (Del.) isolated from different agro-ecological zones in Senegal, and uses of susceptible microbial inoculum in a reafforestation process. METHODS AND RESULTS: A polyphasic approach including phenotypic and genotypic techniques was used to study the diversity and their relationships with other biovars and species of rhizobia. The geographical origins of the strains have limited effect on their phylogenetic and phenotypic classification. Nodulation tests indicated promiscuity of the strains studied, because they were capable of nodulating six woody legume species (Acacia auriculiformis, Acacia senegal, A. seyal, Acacia tortilis ssp. raddiana, Leucaena leucocephala and Prosopis juliflora). Sequencing and phylogenetic analyses of nodA, nodC and nifH genes pointed out that in contrast to nodA gene, the phylogenies of nodC and nifH genes were not consistent with that of 16S rRNA, indicating that these genes of the A. seyal-nodulating rhizobia might have different origins. Microbial inoculation on nonsterile soil had significant effect on the nodules number and the growth of the seedlings, indicating that these strains of rhizobia might be used as inoculum. CONCLUSIONS: The results indicated that A. seyal is a nonselective host that can establish effective symbiosis with Mesorhizobium spp. strains from diverse genomic backgrounds and that the selected A. seyal-nodulating rhizobia could enhance plant growth. SIGNIFICANCE AND IMPACT OF THE STUDY: These results showed the important role that A. seyal could play in the improvement of reafforestation process as a promiscuous host, which can establish effective symbiosis with rhizobia from diverse genomic backgrounds.