The First Evidence of Epidemic Strain Clostridium Difficile (027/NAP1/BI) in Eastern Croatia

A case of the fi rst evidence of epidemic strain Clostridium diffi cile (027/NAP1 (BI) in a patient in Slavonia region (Eastern Croatia) is presented. Clostridium diffi cile infection presents the leading cause of the antibiotic-associated nosocomial diarrhea and colitis in the industrialized world. PCR-ribotype 027 is a hypervirulent strain with great epidemic potential and since 2005 spread to European countries. A study published in 2011 years did not prove the presence of ribotype 027 in Croatia. Case Report The First Evidence of Epidemic Strain Clostridium Diffi cile (027/NAP1/BI) in Eastern Croatia Maja Tomić Paradžik1,3*, Dijana Andrić2, Domagoj Drenjančević3 and Jasminka Talapko3 1Department of Clinical Microbiology, Institute for Public Health Brod-Posavina County, Slavonski Brod, Croatia 2General Hospital Dr Josip Benčević, Slavonski Brod, Croatia 3Faculty of Medicine, University of Osijek, Croatia Dates: Received: 23 February, 2017; Accepted: 02 March, 2017; Published: 04 March, 2017 *Corresponding author: Maja Tomić Paradžik, Department of Clinical Microbiology, Institute for Public Health Brod–Posavina County, Vladimira Nazora 2A, Slavonski Brod 35 000, Croatia, Tel: +385 35 444 051; E-Mail:


Introduction
Clostridium diffi cile is a Gram-positive spore-forming anaerobe that can be found in the stomach and the intestines of healthy people. There are two forms of C. diffi cile bacteria -an active form that cannot survive in the environment for long periods of time and the dominant form, a spore, which can survive for a long period of time. Spores are very diffi cult to remove from surfaces and therefore can contaminate the environment by living on them for weeks to months. Spores cause infection after they have been ingested and have germinated into the active form of C. diffi cile. When the normal fl ora of the intestinal tract is disrupted (e.g. with antibiotics), C. diffi cile can multiply and produce toxins that cause mild to very severe diarrhea known as Clostridium diffi cile infection or CDI [1,2].
Clostridium diffi cile infection is prevalent in health-care facilities and presents the leading cause of the antibioticassociated nosocomial diarrhea and colitis in the industrialized world. Less often, it is acquired in the community from an unknown source. The main risk factors for developing CDI are advanced age, (over 65 years), with comorbidity and the previous use of antibiotics. Administration of the broad-spectrum antibiotics leads to the elimination of the healthy microfl ora of the gut, followed by the loss of colonization resistance, which may allow microbes such as C. diffi cile to colonize, adhere and replicate to levels that cause disease [2,3]. According to the Centre for Disease Control (CDC), 1 in 20 hospitalized patients will acquire a health care-associated infection, and while most health care-associated infections such as methicillin-resistant Staphylococcus aureus are decreasing, C. diffi cile rates continue to rise rapidly [4].
At the beginning of the 21 st century increasing rates of C. diffi cile infection have been reported in Northern Hemisphere, fi rst in the United States of America (USA) and Canada. The prevalence of hospital-acquired Clostridium diffi cile infection (HA-CDI) has recently increased with the spread of the hypervirulent, fl uoroquinolone-resistant strains belonging to the Polymerase Chain Reaction ( PCR) -ribotype 027 [5,6]. Ribotype 027 was the causative agent of the largest C. diffi cile epidemic recorded to date, in which over 2 000 fatalities occurred in Quebec, Canada during 2005. Reports of this outbreak described much higher rates of morbidity and mortality associated with the ribotype 027 than with the other, typical endemic strains (such as ribotype 078). Because of its epidemic potential ribotype 027 is called "hypervirulent" strain. Clear disambiguation between the hypervirulent and typical strains is currently precluded by the incomplete understanding of what causes some strains to generate outbreaks with substantial morbidity [7]. In addition to the usual toxins A and B, these fl uoroquinolone-resistant strains produce a binary toxin.
In Europe, PCR-ribotype 027 was fi rst reported in 2005 in England and shortly after in the Netherlands [8,9] [13].
We report a case of hospital-acquired colitis with diarrhea caused by Clostridium diffi cile PCR-ribotype 027 in a female patient in general hospital in Slavonia, middle-east region of Croatia.
First step in C. diffi cile diagnosis is the detection of C. diffi cile antigen, glutamate dehydrogenase (GDH), by the automated immunoassay system based on the Enzyme Linked Fluorescent Assay (ELFA) principles (mini VIDAS®, BioMérieux, France). This test detects an antigen that is produced in high amounts by C. diffi cile, both the toxin and non-toxin producing strains. Our patient had positive GDH antigen (TV neg 0.10 -TV poz 0.10) with value 7.11.
Positive results are followed by PCR assay Xpert® C. diffi cile (Cepheid, USA). Xpert C. diffi cile /Epi is the fi rst commercially available test in the world for detecting and differentiating the epidemic strain of C. diffi cile (027/NAP1/BI -the strain, which is restriction endonuclease analysis group BI, pulse-fi eld gel electrophoresis type NAP1, and polymerase chain reaction ribotype 027, is designated BI/NAP1/027). This additional testing showed the presence of the toxogenic C. diffi cile and the presumptive positivity of the 027 ribotype.
After the presence of the toxogenic C. diffi cile in the stool had been confi rmed, the patient was moved to a single room at the Department of Infectious Diseases, with the instructions for healthcare workers about the contact isolation and enhanced disinfection of room. Hospital room in which the patient previously resided was disinfected with glutaraldehyde, as well as the room to which she was moved. In the therapy was given oral vancomycin, and patient was required to remain in the single room until she had passed normal stool for 72 hours.

Discussion
C. diffi cile spores are highly desiccation resistant and can persist on hard surfaces for more than 5 months. The accumulation of spores over the bacterial growth cycle demonstrated that hypervirulent strains sporulated earlier and accumulated signifi cantly more spores per total volume of culture than non-hypervirulent strains. Increased rate of sporulation may explain the observation of unusually high relapse rates associated with hypervirulent strains because patients are more likely to contaminate their local environment and subsequently re-infect themselves. Conducted studies have shown three signifi cant differences between the hypervirulent 027 ribotype and other non-hypervirulent strains; hypervirulent strains are more infectious than the epidemic strains; they result in higher rate of symptomatic disease, and outcompete endemic strains in the host's gut [7,15,16]. Due to the dramatic change in the epidemiology of CDI in the last two decades, with the rise of incidence and outbreaks in large and small hospitals, microbiological laboratories must be prepared for these diagnostic challenges, and aware of the limitations of certain diagnostic methods used in previous years. For these reasons, molecular tests in the diagnosis of CDI become necessary.
GDH is considered to be very sensitive, but it is not very specifi c for toxin-producing C. diffi cile. This test indicates if C. diffi cile is present but like bacterial culture do not distinguish toxin-producing from non-toxogenic C. diffi cile isolates. The reason for this approach is that GDH is produced in signifi cantly higher quantities than the C. diffi cile toxin and should yield a more sensitive assay than the solid-phase toxin A/B EIA's. The commercial GDH tests offer a turnaround time of 15-45 min, use it as a screening test to distinguish between the negative and the positive sample, and then go for further processing. Numerous studies have proved that approximately 20 % of the patients who are positive for the GDH antigen of C. diffi cile carry a non-toxogenic strain of C. diffi cile [17]. In our patient, confi rmation test was carried out on Xpert® platform, a very practical and very simple Real-time Polymerase Chain Reaction (RT-PCR) device suitable for small laboratories with a lot of samples and a small number of employees. This is a rapid and very sensitive method for confi rming the presence of the C. diffi cile toxin, but because of the very high cost of the test, it is reasonable to perform the GHD screening test fi rst.
Before PCR assay we used ELFA method (EIA test), but it is not sensitive enough and misses up to 30 % of cases. However, this PCR assay is not recommended for use by some professional organizations [17,18]. ammonium compounds in spore killing has been compared and chlorine-releasing agents are proved to be more effi cient than any other disinfectant for killing spores. But, some previously conducted studies point to the need to apply different types of disinfection depending on the frequency and incidence rates of C. diffi cile in the hospital, so we choose glutaraldehyde for the room disinfection due to low rates of CDI in our hospital, <3.0 cases per 1000 patient-days [19,20]. Hand hygiene which includes washing with soap and water as opposed to alcoholbased hand rubs was recommended during the Intrahospital Infection Control Team (IHI-Team) visit of the department with the CD 027 positive patient. Infection control barriers, antimicrobial stewardship, education of health-care workers and patients can lead to reduction in CDI incidence and decrease in severity, but fi rst step in CDI control is the availability of specifi c and sensitive diagnostic platforms that must be part of every microbiological laboratory today.