| Clostridium difficile (C. difficile) is component of the normal human gut flora and a strictly anaerobic Gram-positive bacillus. C. difficile infection (CDI) refers to infections caused by toxin-producing C. difficile, which is diagnosed with the typical clinical symptoms (usually diarrhea), and positive testing results of C. difficile toxin (toxin-producing C. difficile positive in stool culture, colonoscopic or histopathological pseuolomembranous colitis). Since1978, C. difficile has been considered related with antibiotic-associated diarrhea (AAD). At present, it is appreciated that25%of AAD are caused by the C. difficile. CDI may lead to prolonged hospitalization, increased illness severity and mortality, as well as more medical expenses and burden.PathogenesisC. difficile mainly spreads between people through the fecal-oral route and colonizes in the intestine. Normal intestinal flora can inhibit colonization of C. difficile. While dysbiosis caused by antibiotics easily leads to overgrowth of C. difficile and release of toxin A and B. Toxin A, an enterotoxin, can directly trigger inflammation by affecting intracellular actin in colonic cells. Toxin B, a cytotoxin, can significantly induce cellular apoptosis, resulting in chromatin condensation and cell death. Moreover, C. difficile has cross antigenicity with sordellii, thus C. difficile toxins can be neutralized by sordellii toxin, directly damaging intestinal cells and causing pseudomembranous colitis.Clinical manifestationsMost patients with C. difficile infection present no significant clinical symptoms. Clinical symptoms may manifest as mild to severe watery diarrhea, fulminant colitis and toxic megacolon or obstruction and pseudomembranous colitis. Diarrhea in almost half of the patients occurs during usage of antibiotics or short after discontinuation. Sometimes it develops several months after drug discontinuance. Debilitating diarrhea can occur in severe patients (up to20to30times per day), accompanied by fever, nausea, anorexia, abdominal distension, abdominal pain, etc. Moreover, C. difficile infection often occurs in patients with systemic disease.Auxiliary examinationPatients with C. difficile infection shows thicken colon wall by CT scan. Abdominal X-ray shows paralytic ileus and "thumbprint" syndrome. Colonoscopy shows characteristic changes of colon, where yellow or white oval-shaped patches with clear boundary are scattered, which are strongly adhered with mucosa, and difficult to wash off Punctate pseudomembranous, gray or tan, appear in center of the plaque, blend and uplift in size of a few mm to1cm. There is no significant inflammation between mucosal lesions, but severe lesions might integrate. Decreased albumin levels, elevated levels of white blood cells, blood urea nitrogen, creatinine and serum ferritin levels, dysbiosis of fecal flora and fecal leukocytes may be found.Diagnosis and TreatmentClinical diagnosis of C. difficile-associated diarrhea can be made by the history of broad-spectrum antibiotics, severe diarrhea or hemafecia, and characteristic colonoscopic features (including edema, hyperemia, pseudomembrane, and nonspecific small ulcer or erosion). Confirmed diagnosis depends on results of bacterial culture and toxin test. At present, cytotoxicity assay and toxigenic culture is the standard detection method, but it is time-consuming and requires professional researchers. Therefore, enzyme-linked immunosorbent assay, glutamate dehydrogenase antigen testing and PCR technique are performed in some microbiology laboratories.Metronidazole is the first choice to treat patients with C. difficile-associated diarrhea, taken orally for7to14days (200-250mg,4times per day). The second choice is vancomycin (0.125g,4times per day, for7to14days), available for metronidazole insensitive or critical patients. For patients with severe obstruction, drug can be ingested by the gastric tube or anal tube, and bowel resection may be considered if necessary. Other treatments include bacitracin (25500U,4times per day), teicoplanin (200mg, qd), fusidic acid (500mg, qd), cholestyramine (4g,3-4times per day). Recently, some foreign countries have begun to use fidaxomicin to treat patients who insensitive to vancomycin, and the economic value has been indicated by clinical practice.Relapse of C. difficile-associated diarrhea often occurs7to14days after cure, but there is still no standard treatment for it. Recurrence rate may be reduced by increasing10-14-days’ treatment with metronidazole or vancomycin (125mg, gradually extended dosing interval) in patients with positive C. difficile toxin in stool. For those with multiple recurrences, additional treatment should be considered, such as cholestyramine combined with vancomycin, biological therapy and intravenous infusion of human immunoglobulin.In recent years, more and more attention has been paid to fecal transplantation, as its potential value in recurrent refractory CDI. Faeces are collected from healthy donors (after strict screening for infectious agents), and then transplanted to colon of patients with recurrent CDI in order to restore the patients’ flora homeostasis. The treatment can attenuate the pathological disorders caused by intestinal dysbiosis and is thought to be the most promising treatment. The most commonly used transplantation methods include implantation through enemas, nasal and endoscopic. Numerous studies have shown that it has a good therapeutic effect on recurrent refractory CDI.EpidemiologyOver the past20years, great changes have taken place in the epidemiology of C. difficile. With the wide use of broad-spectrum antimicrobial drugs, incidence of C. difficile-associated diarrhea is keeping rising worldwide. A study in Michigan showed that from2002to2008, rate of CDI has doubled. Recently, C. difficile often outbroke, causing increasing mortality and infection in patients. The outbreak of CDI may be due to the appearing of highly virulent strains (BI/NAP1/027), which characteristic with genetic variation and increased production of toxins, including binary toxin, toxin A and B. The strain is insensitive to fidaxomicin, resistant to quinolone, and has a high recurrence and mortality rate (up to7%). Since the hypervirulent strain debut in Canada, it has been reported in United States, Europe, Japan, Korea, Hong Kong and Singapore. Moreover, infection rates showed an increasing trend in populations such as perinatal women, children and community residences, which were considered with low risk factors.BackgroundCDI is widely reported and monitored in Europe and the United States, while rarely in Asia, except in some developed countries or regions, such as Japan, Korea, Singapore and Taiwan. Report of CDI is not mandatory in medical institutions in Mainland China, Moreover, related researches are limited in some big cities like Beijing and Shanghai due to lack of technology and equipment. Thus, the prevalence of C. difficile in China is not yet clear.AimThe purposes of this study are as follows:①Investigating the prevalence of CDI in Nanfang Hospital, its risk factors and effective surrogate markers, in order to promote rational use of antibiotics, prevent CDI, improve the efficiency of diagnosis and treatment.②Clinically isolating and identifying C. difficile strains, to build a library of C. difficile in Nanfang Hospital, in order to lay the foundation for further study of biological characteristics, pathogenic mechanisms and vaccine development of C. difficile.③Exploring effective and rapid detection of C. difficile, which can be introduced in clinical testing and identification.Materials and MethodsSubjects:A total of4456stool samples were collect from patients in Nanfang Hospital from December2009to December2013(5-7levels in Bristol classification) and analyzed for C. difficile.4132samples were suitable for the study of CDI, of which127cases were a single test specimens,75double repeated samples,33triplicate samples and11four repeat samples.Methods:①Genome DNA were extracted from samples collected from December2009to May2012and used to detect C. difficile toxin B by PCR as previously described by Kato. Clinical data of patients were collected, independent risk factors and surrogate markers of CDI were analyzed by univariate and multivariate logistic regression analysis.②A total of1962samples were inoculated on CCFA anaerobic culture medium (SOP culture) and tested for cell toxicity (cytotoxicity assay, CTA). Typical colonies were separated and purificated, followed by Gram staining, lactate dehydrogenase (glutamate dehydrogenase, GDH) test and biochemical identification. Bacterial DNA were then extracted from the successfully identified C. difficile and tested for A, B toxin, binary toxin detection and ribotyping. All of the identified strains were stored at-80℃and patients’information were saved.③A total of140fresh diarrhea stool samples were collected from adults from2012-07-31to2012-12-19(duplicate samples were excluded) and divided into two parts. One was used for C. difficile standard culture together with biochemical identification and cytotoxicity experiments, and another for Cepheid GeneXpert(?) Dx (Cepheid, GX-XVI) system testing as well. McNemar chi-square was used to compare differences between the above two parts, and parameters including sensitivity, specificity, Youden’s index, positive likelihood ratio etc. were determined as well.ResultsA total of3885samples of diarrhea stool were collected from December2009-May2012in Nanfang Hospital, among which177repeated cases and18cases which did not meet requirement of amount or freshness were excluded, thus a total of3660cases were further used for C. difficile toxin B detection, risk factors and surrogate markers analysis. The mean age of patients was54.9±15.8years old, among which73.3%were male,90.8%inpatients,0.7%ICU patients. Infection rate of C. difficile in Nanfang Hospital was15.6%in patients with diarrhea, and CDI kept in a stable trend. Multivariate logistic regression analysis showed that age, gender, Horn’s index, history of broad-spectrum antibiotics, previous infection, history of cortisol drugs and immunosuppressants, and history of inflammatory bowel disease were all independent risk factors for Clostridium difficile infection, among which, gastric intubation (OR5.759), enema (OR3.976), malignant tumor (OR1.668), antibiotics (OR1.851), cortisol drugs (OR2.223), previous CDI (OR4.107) and inflammatory bowel disease (OR1.894) were more notable. fever (OR1.586) and decreased albumin (OR4.947) can be used as independent markers. In572C. difficile-positive patients,386cases (67.5%) had a history of antibiotic treatment within the past three months. In3088cases of diarrhea specimens without C. difficile, there were1624cases (52.6%) had antibiotic treatment history. Second generation and third generation cephalosporins, clindamycin, fluoroquinolones and carbapenems were all related with the high incidence of CDI.A total of1962fresh stool samples for isolation and identification. The mean age of the corresponding patients was56.46±15.48years old, including1120male patients (57.08%) and842female (42.92%).262samples of C. difficile were obtained, including250toxigenic strains and the positive rate was12.74%. In the included patients,473cases (24.11%) presented with inflammatory bowel disease, including198cases of ulcerative colitis (41.86%) and275cases of Crohn’s disease (58.14%).34cases of CDI occurred in ulcerative colitis and Crohn’s disease groups respectively, and the infection rates were17.17%and12.36%without significant difference (P=0.141). Moreover, no significant difference were observed in gender, including303male cases (64.06%) and170female cases (35.94%)(P=0.878). In addition, in inflammatory bowel disease patients, the average age of C. difficile-positive group was38.35±15.35years old, and negative37.20±14.84years old, without significant difference (P=0.490), indicating that age had no effect on the incidence of C. difficile in inflammatory bowel patients. Our results showed that191samples were A+B+(76.4%) and59A-B+(23.6%). Ten binary toxin positive cases were found in the A+B+strains, and one case of C. difficile027type (BI/NAP1/027) were finally identified. The isolated262cases of C. difficile were numbered, and demographic information of patients including name, ID, time of admission as well as toxin classification, ribotyping results were collected and saved to establish C. difficile library of Nanfang HospitalA total of140fresh diarrhea stool samples were collected in adult patients (>18years) between2012-07-31to2012-12-19, among whom the most common diagnosis was Crohn’s disease (34patients,24.3%), followed by malignant tumors (28patients,20.0%), infectious diseases (23samples,16.4%), chronic disease (18patients,12.9%), ulcerative colitis (15patients,10.7%), post operation (6patients,4.3%), and autoimmune diseases (4patients,2.9%). Forty-eight cases of C. difficile toxin B producing toxigenic strains were obtained by standard SOP culture, cytotoxicity test and neutralization test. Forty-six cases of toxin B-positive were obtained using Cepheid GeneXpert (?) Dx System, without significant difference with golden standard detection. McNemar test showed that sensitivity and specificity of detection using Cepheid GeneXpert (?) Dx System were both more than90%, indicating capacity of identifying infection from uninfection with little missed diagnosis and misdiagnosis. The positive and negative likelihood ratio was42.17and0.085respectively, indicating large possibility of correct detection of C. difficile or identifying uninfected patients. The Youden’s index was89.49, indicating efficiency of CDI detection and higher reliability.ConclusionWe outlined the clinical features, epidemiology and risk factors of C. difficile infection in Nanfang Hospital by epidemiological investigation and risk factor analysis. We concluded that albumin might be the best surrogate marker reflecting changes in CDI progression. Our results also provide important conclusion for how to rationally use antibiotics, prevent CDI, efficiently improve diagnosis and treatment, control hospital epidemic and reduce the incidence and mortality. We successfully established C. difficile bacteria library, which is the foundation of clearly understanding the biological characteristics and pathogenic mechanism of C. difficile, and developing an effective vaccine. More importantly, for the first time, we isolated the highly pathogenic strain027(BI/NAP1/027) in mainland China, revealing the potential outbreak of C. difficile. In addition, our study confirmed that Cepheid Xpert is convenient, rapid and reliable to detect C.difficile. And more importantly, the Cepheid Xpert C.difficile can facilitate binary toxin and027strains detection, and monitor outbreaks of C. difficile. Therefore, Cepheid Xpert can be routinely used in clinical screen of C. difficile. |
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