Current knowledge on the laboratory diagnosis of Clostridium difficile infection

Clostridium difficile (C. difficile) is a spore-forming, toxin-producing, gram-positive anaerobic bacterium that is the principal etiologic agent of antibiotic-associated diarrhea. Infection with C. difficile (CDI) is characterized by diarrhea in clinical syndromes that vary from selflimited to mild or severe. Since its initial recognition as the causative agent of pseudomembranous colitis, C. difficile has spread around the world. CDI is one of the most common healthcare-associated infections and a significant cause of morbidity and mortality among older adult hospitalized patients. Due to extensive antibiotic usage, the number of CDIs has increased. Diagnosis of CDI is often difficult and has a substantial impact on the management of patients with the disease, mainly with regards to antibiotic management. The diagnosis of CDI is primarily based on the clinical signs and symptoms and is only confirmed by laboratory testing. Despite the high burden of CDI and the increasing interest in the disease, episodes of CDI are often misdiagnosed. The reasons for misdiagnosis are the lack of clinical suspicion or the use of inappropriate tests. The proper diagnosis of CDI reduces transmission, prevents inadequate or unnecessary treatments, and assures best antibiotic treatment. We review the options for the laboratory diagnosis of CDI within the settings of the most accepted guidelines for CDI diagnosis, treatment, and prevention of CDI.

Cholera:a great global concern

Cholera,caused by the infection of toxigenic Vibrio cholerae(V.cholerae) to humans,is a life threatening diarrheal disease with epidemic and pandemic potential.The V.cholerae,both 01 and 0139 serogroups,produce a potent enterotoxin(cholera toxin) responsible for the lethal symptoms of the disease.The O1 serogroup has two biotypes(phenotypes),classical and El Tor;each of which has two major serotypes(based on antigenic responses),Ogawa and Inaba and the extremely rare Hikojima.V.cholerae O1 strains interconvert and switch between the Ogawa and Inaba serotypes.Fluid and electrolyte replacement is the mainstay of treatment of cholera patients;the severe cases require antibiotic treatment to reduce the duration of illness and replacement of fluid intake.The antibiotic therapy cunenlly has faced difficulties due to the rapid emergence and spread o(multidrug resistant V.cholerae causing several outbreaks in the globe.Currently,cholera has been becoming endemic in an increasing number of geographical areas,reflecting a failure in implementation of control measures.However,the current safe oral vaccines lower the number of resistant infections and could thus represent an effective intervention measure to control antibiotic resistance in cholera.Overall,the priorities for cholera control remain public health interventions through improved drinking water,sanitation, surveillance and access to health care facilities,and lurther development of safe,effective and appropriate vaccines.Thus,this review describes the facts and phenomena related to the disease cholera,which is still a great threat mainly to the developing countries,and hence a grave global concern too.

Detection of toxigenic Clostridium difficile by polymerase chain reaction

The polymerase chain reaction (PCR) was used for the detection of toxigenic Clostridium difficile (Cd). A primer pair derived from non-repeating sequences of the toxin A gene were used to amplify a 306 bp DNA fragment. Amplified products were visualized by polyacrylamide gel electrophoresis followed by ethidium bromide staining. All 19 strains of toxigenic Cd generated single specific amplified DNA. In contrast, none of the 8 strains of non-toxigenic Cd, 2 strains of C. sordelli and 2 strains of E. coli gave positive results. After the detected DNA of toxigenic Cd was diluted to 0.5ng, the polymerase chain reaction assays were still positive. The results demonstrate that polymerase chain reaction is a simple, rapid, specific and sensitive method for the detection of toxigenic Cd and could be used for the direct detection of Cd in feces samples.

Effect of Robusta (<i>Coffea canephora P.</i>) Coffee Cherries Storage after Harvest before Putting Out for Sun Drying on Development of Toxigenic Fungi and the Variation of the Physicochemical Components

In this study, the effects of the storage duration of coffee cherries after harvest before putting out for sun drying on the kinetics of drying, fungi development and the variation of physicochemical content were evaluated. The results showed that the longer coffee cherries were stored after harvest before putting out for sun drying, the quicker they dried. Indeed, the drying durations were 19, 16, 12, 10, 7 days respectively for coffee cherries put out for sun drying at the day of harvest, the second, the fourth, the sixth and the eighth day after harvest. However, this storage of the cherries after harvest before putting out for sun drying led to the increasing to the infection of cherries by fungi. Indeed, samples of more contaminated inside were those from the lots of cherries stored 8 days after harvest before putting out for sun drying with 55.55% of the samples infected with a percentage of infected beans between 10% and 50%, and 44.45% of the samples were infected with a percentage of infected beans between 50% and 100%. Furthermore, those put out for sun drying at the day of harvest were free inside by fungi. Among the fungi isolated, toxigenic species was found. However, no relationship between the frequencies of ochratoxin A producing strains isolated and the storage duration of the cherries after harvest before putting out for sun drying was noted. This storage of the cherries after harvest before putting out for sun drying also led to the acidification of the cherries (pH = 5.27 - 3.6) and the degradation of their chlorogenic acids content (12.58% - 10.30%) while for their caffeine content (2.53% - 2.55%). No significant difference was observed about the storage duration of the cherries after harvest before putting out for sun drying.

Toxigenic Fungi and Mycotoxins in Organic Spelt and Its Products

This study shows that the main cause of Fusarium head blight of spelt was F. poae. In 2007 deoxynivalenol was found up to 0.27 mg/kg in 2 of 18 samples of winter spelt kernels from organic farms. Also in 3 samples T-2 toxin was found in amount below 0.075 mg/kg. Aflatoxins and ochratoxin A were not found in kernels. Among nine of the examined samples of winter spelt in 2008, DON was identified in all samples （up to 0.31 mg/kg）, while T-2 toxin, aflatoxins and OTA were not found. Among twenty of the examined cultivars of winter spelt, deoxynivalenol was identified in 6 samples （up to 0.3 mg/kg）, T-2 toxin was identified in one sample in very low amount （below 75 μg/kg） while aflatoxins and ochratoxin A were not found. Deoxynivalenol was found in following winter spelt cultivars： T. spelta L. album, T. spelta BG, T. spelta BG 1166, T. spelta, Schwabenspelz and Franckenkorn. T-2 toxin was identified in T. spelta L. album BG 31. Among 13 products from spelt, DON was detected in 1 sample, OTA in 1 sample and zearalenone in 1 sample, T-2 toxins and aflatoxins were not found.

Fungal Population Dynamics in Ready-to-eat Salads During a Shelf-life in Italy

The aim of this work was to investigate the fungal population dynamics in ready-to-eat bagged samples of rocket （Diplotaxis spp.）, lettuce baby leaf （Lactuca sativa L.） and ＂songino＂ （Valerianella olitoria L.） during a shelf-life, in order to evaluate the effects of the storage length and season of production on the spoilage processes. The incidence of toxigenic moulds was particularity studied in order to evaluate a potential production of mycotoxins and allergenic conidia. A total of 900 samples collected from 10 Italian trademarks were analyzed at the 2nd, 5th and 8th day after the packaging in the spring and summer. A very high number of fungi was found and a great variability of moulds and yeasts at the 1 st day of sampling was observed. Regarding to season of production, any seasonal effect on the moulds and yeasts has been observed, but the moulds detected belonged to different species in relation to season. Regarding to storage length, the yeasts and moulds did not showed significant variations during a shelf-life. In relation to vegetable species, the lettuce resulted always less contaminated with respect to other salads, and the rocket presented 1-2 Log cfu/g of increasing in the level of moulds. Regarding to fungi species, the yeasts were significantly predominant respect to moulds. Finally, the toxigenic moulds Aspergillusflavus and Penicillium italicum were found in all the types of salad in the summer, and their growth during the storage at low temperature represented a potential hazard for the mycotoxins and allergenic conidia production in these commodities.

A Preliminary Molecular Typing by PCR Assays of <i>Clostridium perfringens</i>and <i>Clostridium difficile</i>Isolates from Dogs

Clostridium perfringens and C. difficile have been associated with acute and chronic large and small bowel diarrhoea, and acute haemorrhagic diarrhoeal syndrome in dogs. The objective of this study was to investigate by toxin gene profile and PCR-ribotyping the molecular characteristics of 14 C. perfringens and 10 C. difficile isolates from 95 canine faeces (n = 36, diarrhoeic and n = 59, non-diarrhoeic). Concerning C. perfringens, 13 strains (92.9%) were type A, of which 3 (23.1%) also possessed the beta 2 toxin (CPB2)-encoding gene. One isolate (7.1%) was type D and possessed CPB2 gene. On the whole, 4 of the 14 strains (28.6%) tested cpb2-positive. Six C. difficile isolates (60.0%) demonstrated tcdA+/tcdB+ and cdtA+/vcdtB+ genotype and tested positive for, in vitro, toxin production by EIA. Eight distinct ribotypes were observed. In conclusion, the PCR assays may provide useful and reliable tools for C. perfringens and C. difficile molecular typing in routine veterinary diagnostics.

The Nature, Sources, Detections and Regulations of Mycotoxins That Contaminate Foods and Feeds Causing Health Hazards for Both Human and Animals

Mycotoxins are toxic secondary metabolites produced by fungus kingdom. Fungi (molds) under aerobic and optimum conditions of humidity and temperature consume nutrients for proliferation and mycotoxin production (secretion). There are seven major groups of mycotoxins produced by different species of toxigenic fungal genus. Mycotoxins production from these toxigenic fungi depends on the surrounding intrinsic and extrinsic environments. These seven mycotoxins groups that contaminate grains, foods and animal feeds are: Aflatoxins, Trichothecene, Ochratoxins, Ergot alkaloid (Ergolin), Fumonisins, Patulin, and Zearalenone. These mycotoxins are capable of causing health hazards and death for both human and animals by effecting mammalian cells, causing a number of problems in normal cell function and a wide variety of clinical symptoms of diseases. These mycotoxins are varied in their toxicity depending on the infected host (human or animal) and the host susceptibility (immunity). The major concern of food and feed industries is the contamination of food products and animal feed supplies by these mycotoxins. Worldwide Health Organization (WHO), and Food and Agriculture Organization (FAO) are responsible to regulate the acceptable (tolerable) levels of these mycotoxins in grains, food and feed supplies to ensure the safety and health for both human and animals. Understanding fungal ecology and factors that affect fungal proliferation and mycotoxins production by these toxigenic fungi in agriculture crops as raw materials for both human food and animal feed products, plus understanding the chemistry and property of these mycotoxins, methods of detection, illness symptoms, and comply with regulatory guidance established by World Health Organization (WHO)/Food and Agriculture Organization (FAO) are key factors to prevent or minimize foods/feeds contamination and the toxicity of these mycotoxins for both human and animals health, plus reducing economical loss.

Molecular and Susceptibility Analysis of Toxigenic <i>Clostridium difficile</i>Obtained from Adult Patients Suspected of CDI in Trinidad

Clostridium difficile is a Gram positive rod-shaped bacterium that produces two major toxins, A and B. The detection of the organism and its toxins has been widely carried out using specialized Enzyme-Linked Immunosorbent Assay (ELISA) kits;however these generally have been unsuccessful in identifying all Clostridium difficile positive samples. In this study, fifteen clinically symptomatic patients from three of the five major regional hospitals in Trinidad were investigated for Clostridium difficile infections. Stool samples were assessed by ELISA and cultured isolates were characterized using agar dilution antibiotic sensitivity assays, conventional Polymerase Chain Reaction (PCR), DNA sequencing and phylogenetic analysis of toxin A and B genes. All 15 patient stool samples and isolates were positive for toxigenic Clostridium difficile via ELISA and PCR respectively. All isolates were positive for the housekeeping tpi and Toxin B genes by PCR but only three of these were positive for the Toxin A gene. The Toxin B gene sequences showed 100% similarity levels among isolates while the Toxin A gene sequences showed 99% similarity among isolates. Phylogenetic analysis showed that the strains isolated in Trinidad most likely belonged to the same strain/group. Agar dilution sensitivity tests showed highest susceptibility to Pipercillin/Tazobactam and Meropenem (87%) and the highest resistance was seen with Cefotaxime in 93%. These results indicate that similar virulent strains of C. difficile are present in the Trinidad population and that pathogenic strains are more likely to be susceptible to Pipercillin/Tazobactam and Meropenem.

Characteristic of Species Composition of Fungi Involved in the Formation of Mycobiota of Honey Bees in Azerbaijan

The purpose of the presented work was dedicated to identifying the species composition of the mycobiota of honey bees in Azerbaijan condition. From the samples taken from bees, materials became clear that in the formation of mycobiota those materials (from bees, from where they live and their products) in generally participate 52 species of fungi. Among the recorded fungi, species take part such as Alternaria alternata (Fr.) Keissl., Aspergillus flavus Link, Candida albicans (C.P. Robin) Berkhout, Cladosporium herbarum (Pers.) Link, Penicillium cuclopium Westling, P. granulatum Bainier and etc. which carry features conditionally pathogenicity, toxicity, allergens and danger to biological productivity of bees and as well as to pollution of their products. It is known for a long time to scientists that these species are dangerous for human health. For this reason, preparation of normative documents that reflect the principles of microbiological safety of bee products is a necessary task.

Pollution of Airborne Fungi in Naturally Ventilated Repositories of the Provincial Historical Archive of Santiago de Cuba (Cuba)

Environmental fungi can damage the documentary heritage conserved in archives and affect the personnel’s health if their concentrations,thermo-hygrometric parameters and ventilation conditions are not adequate,problems that can be accentuated by Climate Change.The aims of this work were to identify and to characterize the airborne fungal pollution of naturally ventilated repositories in the Provincial Historical Archive of Santiago de Cuba and predict the risk that these fungi pose to the staff’s health.Indoor air of three repositories of this archive and the outdoor air were sampled in an occasion every time in 2015,2016 and 2017 using a SAS sampler.The obtained fungal concentrations varied from 135.6 CFU/m^(3) to 421.1 CFU/m^(3) and the indoor/outdoor ratios fluctuated from 0.7 to 4.2,evidencing a variable environmental quality over time,but in the third sampling the repositories environments showed good quality.Aspergillus and Cladosporium were the predominant genera in these environments.A.flavus was a prevailed species in indoor air,while A.niger and Cl.cladosporioides were the species that showed the greatest similarities with the outdoor air.Coremiella and Talaromyces genera as well as the species Aspergillus uvarum,Alternaria ricini and Cladosporium staurophorum were the first findings for environments of Cuban archives.Xerophilic species(A.flavus,A.niger,A.ochraceus,A.ustus)indicators of moisture problems in the repositories were detected;they are also opportunistic pathogens and toxigenic species but their concentrations were higher than the recommended,demonstrating the potential risk to which the archive personnel is exposed in a circumstantial way.

Detection of antibiotic resistance toxigenic Clostridium difficile in processed retail lettuce

Objectives:Clostridium difficile is the major cause of infectious diarrhoea in humans after antimicrobial treatment.Clostridium difficile has been isolated from food animals and meat.The main purpose of this study was to characterize C.difficile isolated from retail lettuce and determine the antibiotic resistance using five common clinical-selected antibiotics(metronidazole,vancomycin,clindamycin,erythromycin,and cefotaxime).Materials and Methods:Lettuce samples(grown in California,Arkansas,and Louisiana)were purchased from retail stores.Results:Toxigenic C.difficile was isolated from 13.8 per cent(41/297)of the lettuce samples.Among the toxigenic isolates,only 82.9 per cent(34/41)produced toxin B,17.1 per cent(7/41)produced both toxin A and toxin B,and two of the Louisiana C.difficile isolates were identified as ribotype 027.Under the treatment of the five antibiotics,the virulence C.difficile isolates were identified as having antibiotic resistance to metronidazole,vancomycin,and erythromycin.Conclusion:The present study reports the highest prevalence of toxigenic C.difficile in US retail lettuce.The antibiotic resistance to metronidazole,vancomycin,and erythromycin of the isolated C.difficile from retail lettuces could lead to public health concerns.

The study of ctx B and rstR variations of toxigenic Vibrio cholerae O1 E1 Tor strains isolated from 1961 to 2010 in China

Objective To understand the ctx B and rstR variations of toxigenic Vibrio cholerae(V.cholerae)O1 E1 Tor strains isolated from different provinces in China from1961 to 2010.Methods All 385 toxigenic V.cholerae O1 E1 Tor strains were selected,which were isolated in China between year 1961 and 2010.ctx B gene was amplified by PCR method and sequenced for further analysis.rstR was detected with PCR by using the

Phenotypic diversity of toxigenic Vibrio cholerae O1 E1 Tor strains identified in China

Objective To understand the phenotypic diversity of toxigenic Vibrio cholerae O1 E1 Tor strains isolated from different provinces in China during the last 50 years.Methods Traditional biotyping testings including susceptibility to polymyxin B,sensitivity to groupⅣphage,Voges-Proskauer test and haemolysis of sheep erythrocytes were conducted.Results Data from Biotype-specific phenotype analysis revealed that only 133 isolates carried the typical E1 Tor phenotypes while the other