Clostridium difficile - Culture; cytotoxin detection in cell culture; detection of toxin genes A, B and binary toxin by PCR; detection of gene deletions in tcdC (hypervirulent strains); Ribotyping identification (hypervirulent strains NAP1 / BI / 027 and others).

 

Information (14/04/10)

 

Clostridium difficile, an anaerobic spore strict production, was recognized as a potential pathogen enteric late 1970s, when it was related to cases of pseudomembranous colitis by administering appearing broad spectrum antimicrobials.

Subsequently it has been found that its pathogenicity is related to the 20 to 30% of patients presenting diarrheas associated with antibiotics; with 50 to 75% of cases of antibiotic - associated colitis, and over 90% of cases of patients with pseudomembranous colitis. This is because Clostridium difficile can produce one or two types of toxin (A - a enterotoxina-, and toxin B - a citotoxina-). These toxins cause diarrhea (enterotoxin), or severe inflammation of the colon (cytotoxin). Most people affected are elderly, hospitalized or admitted to homes for the chronically ill old, who after being treated with antibiotics developed diarrhea or antibiotic - associated colitis. However, currently they are occurring cases of diarrhea and colitis associated with Clostridium difficile, in people who were not considered at risk such as young individuals, or which have not been treated with antibiotics, or have been admitted to hospital.

Most pathogenic strains produce toxin A (an enterotoxin) and toxin B (a cytotoxin) (A-positive strains toxin, toxin B-positive = A + B +). They have also found toxin producing strains B only (A-negative staphylococcal strains; toxin B-A-B = positive +). Toxins A and B are similar to other clostridial cytotoxins glycosyltransferases.

The two aforementioned A and B toxins are encoded by two distinct genes: tcdA and tcdB, together with tcdC, TCDD and TCDE genes form a locus of 19.6 kb pathogenicity (SWAP locus with the following gene order: TCDD gene, gene tcdB, TCDE gene, tcdA gene tcdC gene). These toxins are responsible for the intestinal damage that occurs during infection. Strains of C. difficile produce toxins that are not nonpathogenic. Therefore, to make a correct diagnosis should be investigated for the presence of one or both toxins in stool samples. Isolation of toxigenic strains (toxigenic crops) contributes to the diagnosis of C. difficile associated diarrhea.

Toxigenic typing differentiates variants A-B +, where the 3'end of the toxin gene has a deletion and differentiate strains A + B +. It was thought that variants A-B + were not pathogenic and were isolated from asymptomatic children. However, several authors have reported cases of pseudomembranous colitis, or diarrhea associated with antibiotics, by strains A-B +.

Strains A-B + were detected in 1991-1992 (CCUG8864). These strains had a deletion in the gene for toxin A and yet cause disease in the animal model. Toxin B of these strains is weakly enterotoxic in the experimental model of the intestinal loop of rabbits and 10 times more lethal than other toxins B. In 1993, it was observed that serotype F of other variants A-B + which was avirulent in animal model it is usually isolated from asymptomatic children. Once available crop can detect an internal gene fragment triosephosphate isomerase (tpi: triose phosphate isomerase) which is a housekeeping gene, an internal fragment of toxin gene B (tcdB), and region 3 ', deleted or not, the toxin gene a (tcdA). 10 toxinotipos have been characterized (I to X), according to deletions or additions within various regions of the toxin genes or other regions of toxigenic element SWAP (pathogenicity locus).

In 2002, he appeared an epidemic strain of Clostridium difficile (strain NAPI / BI / 027) characterized by the process involve more serious. This strain has a deletion of a nucleotide (e.g. nucleotide 117;. TcdC ?117), or sometimes up to 18 bp (?330-347) in gene regulatory tcdC resulting in overproduction of toxins A and B since that gene involved in regulating the expression of the toxin, but also some of these strains are producing binary toxin. These strains are resistant to antimicrobials. The strain produced epidemic outbreaks in the US and some countries in Europe. Binary toxin found in these strains is different in activity to the toxin A or B (glycosyltransferases) and having an ADP-ribosyltransferases specific actin (Actin-specific ADP-ribosyltransferase), similar to the toxin "t" Clostridium perfringens.

The NAP1 / BI / 027 strain is one of the hypervirulent strains resistant to fluoroquinolones and other antimicrobials such as macrolides. This strain is named for NAP1: North American Pulse field gel electrophoresis type 1-PFGE (type 1 Electoforesis of pulsed fields in North America); BI distinct type restriction enzyme -Restriction Endonuclease Analysis-; 027 by the type obtained by ribotyping Ribotyping- -PCR. This strain is often called sometimes only as "Ribotype 027" or just "strain 027" since typing by PFGE usually not performed, nor the typing restriction endonuclease. Today there are other ribotype also correspond to hypervirulent and resistant strains are spreading, even surpassing Ribotype 027, such as 037, 018 and 078.

Epidemiology

This bacterium is found in the environment (soil, water, human waste and animal foods of various types, ...), and many people are intestinal carriers of it. Health centers exist in larger number of carriers in the intestine. The spores of this bacteria are excreted with the feces of humans and animals, and can remain in the environment for weeks or months, contaminating food and objects when there is no good hand hygiene. The prevalence of intestinal colonization may be 50% in hospitalized patients, 5 to 7% by Residents of longer stay, and 2% of adult outpatients. Among hospitalized carriers, the rate of colonization is higher in patients who received antibiotics.  

Risk factor's

Risk factors are considered:

• Administration current or recent antimicrobial broad spectrum of several simultaneously or over an extended period.

• Stays at health centers and the like: to be, or have been, in a health center, nursing homes, Center for chronically ill. These centers bacteria spreads easily by frequent use of antimicrobials, to exist in them more vulnerable, which facilitates the spread-.persona contact person, and through surfaces or instruments (tables beds, toilets, toilets , stethoscope, thermometers, telephones, ...).

• Serious illness or medical procedure: inflammatory bowel disease, colorectal cancer, immunosuppression, cancer chemotherapy, abdominal surgery, ...

Pathogenesis

In the intestine there many bacteria (107 aerobic/g and 109 anaerobics/g stool). These bacteria defend us from infections, while we provide metabolic cofactors that we absorb. Clostridium difficile infection is established in three steps: 1) disruption of normal intestinal flora following the administration of antibiotics. Clindamycin was the first antibiotic related pseudomembranous colitis; then almost all antimicrobials have been associated, cephalosporins, penicillins and fluoroquinolones. When administering an antimicrobial to treat an infection person, they destroy a part of the intestinal bacteria and leave a free habitat for Clostridium difficile proliferates; 2) acquisition of a toxigenic strain of Clostridium difficile. The bacteria can be found in hospitals, where while there is greater potential for contamination. In many cases, transmission occurs by health workers through contaminated hands; 3) Once established in the gut, it produces / s toxin / s. The exact incubation period is unknown, but is usually not more than 7 days. The / s toxin / s made / s produce watery diarrhea, and destroy the cells of the mucosa in the colon triggering colitis.  

Symptoms

Some people are carriers of Clostridium difficile in the gut but never get sick, even if they can spread the infection. Clostridium difficile disease usually develop during treatment with antimicrobial, or even after a few months after being treated with antibiotics.

Most patients have mild or moderate infection, the most frequent watery diarrhea, liquid stools with three or more times a day, for 2 or more days. On other occasions they have severe infection, accompanied by dehydration requiring hospitalization due to an inflammation of the colon with ulcerated areas that bleed and fester (pseudomembranous colitis). In these cases, patients have watery diarrhea, with 10 or 15 daily stools, abdominal pain, fever, blood and pus in stool, nausea, dehydration, anorexia, weight loss, abdominal distension, leukocytosis, and renal failure. 

Complications:

Dehydration, renal failure, toxic megacolon (distention gases produced by the bacteria), intestinal perforation (by extensive damage to mucosa), death.

Treatment:

Is recommended to stop the administration of antimicrobial being administered, and if required his administration, switch to another.

The most recommended antimicrobials are:

• Oral metronidazole (500 mg po tid for 10 days in severe cases iv 500 mg q6h along with vancomycin -see below-). It is the antimicrobial of choice for mild to moderate cases of infection. It is as effective as vancomycin, although some raise doubts about the effectiveness identical between the two. In patients who can not take oral medication, or have intense ileus may be administered intravenous metronidazole, vancomycin intracolonic with nasogastric. Metronidazole be used as first choice to avoid the risk of selection of Enterococcus spp. Vancomycin - resistant. No resistance to metronidazole are known.

• Vancomycin (125-250 mg po qid for 10 days in severe cases 500 mg po Q6d, and enema 500 mg q6h). It should be reserved for patients who can not tolerate metronidazole, which do not respond to metronidazole, or have a serious infection. It is recommended as an alternative to avoid the risk of selecting Enterococcus spp. Vancomycin - resistant. No resistance to vancomycin is known.

• Fidaxomicin. It has been approved for this use and is considered as effective for treating mild to moderate infections, and appears to decrease the risk of recurrence by strains than NAPI / BI / 027.

Other adjuvants measures are:

• Administration of probiotics: bacteria and yeast to restore the balance of intestinal flora. In this regard, Saccharomyces boulardii has been used together with antibiotics to prevent recurrences. In Asia (Japan, Korea, China), Clostridium butyricum Miyairi strain 588 (0.35 x 10⁶ CFU) used for interfering with the growth of Clostridium difficile.

• Faecal microbiota transplantation (Transplant feces) are being conducted clinical trials with this procedure (FMT: Fecal microbiota Transplant).

Laboratory diagnosis

There are several methods for detecting the presence of stool toxin and for characterizing the strains, we are set forth below:

• Enzyme immunoassay method (ELISA): This method is rapid (2-6 hours), but is less sensitive (sensitivity 65-85%, specificity: 95-100%), less sensitive to the cytotoxicity test, and provides a rate high false negative. There enzyme immunoassay methods to detect toxin A, or both A and B together. These methods may be performed with or stool filtrates filtered toxigenic crops.

• Cell cytotoxicity method: This method is highly sensitive and specific (sensitivity 80-90%, specificity: 99-100%), but it is slow because it requires use cell cultures to which a filtering feces is added, and It does not allow to have the results within 24 or 48 hours. Moreover, only toxin B detects (cytotoxin). To detect B toxin - producing strains is the reference standard (Gold Standard). However, this test is not commonly performed in clinical microbiology laboratories because it requires the use of cell cultures and confirmation using specific antitoxin. Crops grown from feces in which the toxin is detected (toxigenic crops) has been shown to be as sensitive as cytotoxicity assays and sometimes detect toxigenic strains in samples test negative cytotoxicity, even in patients with clinical evidence of disease associated with C. difficile. In addition, stool cultures allow for adequate insulated toxigenic typing.

• Stool culture highly sensitive and specific (sensitivity: 90-100%, specificity: 98-100%), and allows strain typing case of outbreaks. It Requires 2 to 5 days for completion, and is not specific to toxigenic strains, so it needs to be completed with a test cell cytotoxicity.

• PCR method (polymerase chain reaction): This method is highly sensitive and specific (sensitivity 92-97%, specificity: 100%), and quite fast. These methods allow to know the presence or absence in strains of genes encoding toxin A, toxin B, or binary toxin.

• Identification of strains: amplification methods, followed by genomic sequencing allow to know the existence of deletions, such as those occurring in the NAP1 strains, and identify its ribotyping (eg 027 or other).

• Typed MLST (multilocus Sequence Typing). This genetic typing would be necessary if we wanted to compare strains from different patients, or to compare strains in a patient who had a recurrence, to see if it is a recurrence of relapse-reactivation with the same strain of the patient, or differentiate if it has been re - infection with a different strain.

Tests in IVAMI:

• Isolation of Clostridium difficile in culture and detection of toxin producing strains by PCR.

• Detection of toxins from feces directly by inoculating filtered faeces in cell cultures (cell cytotoxicity). To detect the production of cytotoxins also we have test cell culture cytotoxicity. This test is slower than the molecular detection producers toxin genes obtained by PCR and the same conclusion, although is considered the reference standard (Gold Standard).

• Detection of toxin producing bacteria directly in stool by PCR to detect and tcdB tcdA genes coding for toxins. This is proof that we recommend for its speed, sensitivity and specificity. To detect strains producing toxins A and B, as well as the binary toxin possessing some of the hypervirulent strains, such as NAP1 / BI / 027, by PCR. An alternative to the detection of the binary toxin for hypervirulent strains is detecting genetic deletions in the regulatory gene "tcdC" since one of the characteristics of the hypervirulent strains is that they lack one or more nucleotides in this regulatory gene, so which can not control the expression of toxins a and / or B. analysis of gene tcdC is performed by PCR followed by sequencing to see if has deletions.

• T ipado strains by ribotyping or MLST. We can detect any ribotyping strains by amplification and sequencing of 16S-23S intergenic (strains 027 or other) region. Also we can make the MLST test to compare each other strains from different patients, or strains obtained from the same patient in cases of recurrence.  

Recommended sample:

• Diarrhea, watery stools. It is not recommended to perform a test for Clostridium difficile toxin if stools are not diarrhea or liquid.

Preservation and shipment of sample:

• Cooled: 24 to 48 hours.

• Frozen: more than 48 hours.

Delivery time :

• Molecular methods (PCR only): 24-48 hours;

• Molecular methods (PCR) with sequencing (ribotyping, tcdC gene): 4-5 days;

• Toxin cell culture: 7 days.

Cost of the test:

• Consult ivami@ivami.com