International Journal of Antimicrobial Agents 45 (2015) 1–7

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Review

Clostridium difficile infection in Thailand Papanin Putsathit a , Pattarachai Kiratisin b , Puriya Ngamwongsatit c , Thomas V. Riley a,d,∗ a

Microbiology & Immunology, School of Pathology & Laboratory Medicine, The University of Western Australia, Crawley, WA 6009, Australia Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand c Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73170, Thailand d Department of Microbiology, PathWest Laboratory Medicine, Queen Elizabeth II Medical Centre, Nedlands, WA 6009, Australia b

a r t i c l e

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Article history: Received 22 September 2014 Accepted 25 September 2014 Keywords: Clostridium difficile Epidemiology Thailand Asia Developing countries Antimicrobials

a b s t r a c t Clostridium difficile is the aetiological agent in ca. 20% of cases of antimicrobial-associated diarrhoea in hospitalised adults. Diseases caused by this organism range from mild diarrhoea to occasional fatal pseudomembranous colitis. The epidemiology of C. difficile infection (CDI) has changed notably in the past decade, following epidemics in the early 2000s of PCR ribotype (RT) 027 infection in North America and Europe, where there was an increase in disease severity and mortality. Another major event has been the emergence of RT 078, initially as the predominant ribotype in production animals in the USA and Europe, and then in humans in Europe. Although there have been numerous investigations of the epidemiology of CDI in North America and Europe, limited studies have been undertaken elsewhere, particularly in Asia. Antimicrobial exposure remains the major risk factor for CDI. Given the high prevalence of indiscriminate and inappropriate use of antimicrobials in Asia, it is conceivable that CDI is relatively common among humans and animals. This review describes the level of knowledge in Thailand regarding C. difficile detection methods, prevalence and antimicrobial susceptibility profile, as well as the clinical features of, treatment options for and outcomes of the disease. In addition, antimicrobial usage in livestock in Thailand will be reviewed. A literature search yielded 18 studies mentioning C. difficile in Thailand, a greater number than from any other Asian country. It is possible that the situation in Thailand in relation to CDI may mirror the situation in other developing Asians countries. © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

1. Introduction Clostridium difficile is a Gram-positive, anaerobic, spore-forming bacillus, the most common cause of antimicrobial-associated infectious diarrhoea in hospitalised adults in the developed world [1]. Exposure to this organism may lead to asymptomatic gastrointestinal tract infection, but it can also lead to symptoms ranging from mild diarrhoea to severe colitis and, rarely, to pseudomembranous colitis (PMC), toxic megacolon, intestinal perforation, sepsis and death [2]. In addition to its role as a human pathogen, C. difficile is also found commonly in the gastrointestinal tracts both of symptomatic and asymptomatic animals. This gave rise to speculation that animals may act as a reservoir of human infection [3]. Although there have been numerous investigations of the epidemiology of C. difficile infection (CDI) in North America and Europe, limited studies

∗ Corresponding author. Present address: Microbiology & Immunology, School of Pathology & Laboratory Medicine, Queen Elizabeth II Medical Centre, Nedlands, WA 6009, Australia. Tel.: +61 8 6383 4355; fax: +61 8 9382 8046. E-mail address: [email protected] (T.V. Riley).

have been undertaken elsewhere, particularly in Asia. Interestingly, there are several publications regarding CDI in humans in Thailand, however no work has been published on animals. This is despite the high prevalence of antimicrobial usage in Thai livestock and the fact that antimicrobial exposure is the major risk factor for the acquisition of C. difficile [1]. Given the poorly controlled use of antimicrobials in this country, it is conceivable that CDI is relatively common both in humans and animals. The main purpose of this review was to describe current knowledge of CDI in Thailand. It will address recent trends in CDI epidemiology as well as antimicrobial usage in livestock and pig farming in relation to C. difficile transmission. It is possible that the picture in Thailand in relation to CDI may mirror the situation in other developing Asians countries. 2. Historical perspective and changing epidemiology C. difficile was first described as part of the microflora of neonatal meconium by Hall and O’Toole in 1935. The name Bacillus difficilis was chosen to reflect its morphology and difficulty in culturing [4]. Although its toxigenic potential was known early, it was not until 1978 that the association of C. difficile with PMC was first recognised

http://dx.doi.org/10.1016/j.ijantimicag.2014.09.005 0924-8579/© 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

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[5]. Following this milestone, disease caused by C. difficile remained largely underappreciated until the early 2000s when interest in CDI increased owing to dramatic changes in CDI epidemiology. The first of these resulted from the emergence of the PCR ribotype (RT) 027 strain of C. difficile in North America. This strain caused epidemics initially in Canada and the USA, and later in Europe [6,7]. Whole-genome sequencing and phylogenetic analysis of a global RT 027 collection revealed two lineages, both of which had independently acquired an identical mutation in the DNA gyrase subunit A gene leading to fluoroquinolone resistance [6]. Disease associated with epidemic RT 027 C. difficile exhibited increased morbidity and mortality, initially thought to be due to an increase in toxin A and B production caused by an 18-bp deletion in tcdC, the putative negative regulator for tcdA and tcdB [8,9], but later shown to be specifically caused by a point mutation at position 117 [10]. A second important event has been the emergence of RT 078 as a significant cause of human infection. In 2007, RT 078 was the predominant strain in piglets and calves in the USA [11]. In a multinational study involving 34 European countries, RT 078 was the third most common ribotype causing disease in hospital patients in Europe [12]. A recent study from The Netherlands reported no single nucleotide polymorphism differences when whole-genome sequencing data on RT 078 strains collected from a pig farmer and pigs were compared [13]. As with RT 027, RT 078 produces binary toxin, but has a 39-bp tcdC deletion and a point mutation at position 184 that also leads to greater toxin A and B production [14]. In addition, in contrast to infection caused by RT 027, which is largely healthcare-associated and affects older (>65 years) people, RT 078 is often associated with community-acquired cases and affects previously low-risk, younger individuals [14,15]. Human isolations of RT 078 in the USA have risen also [16,17]. Such findings highlight the zoonotic potential of C. difficile and the possible role that animals may play as a reservoir of infection outside healthcare facilities. In response to these recent events, an increasing number of reports investigating CDI in North America and Europe has been published, however studies in Asia remain limited. 3. Epidemiology of C. difficile in Asia RT 027, which is still the major ribotype in North America [18], has been reported only sporadically in Hong Kong, Japan, South Korea, Singapore and, more recently, China [19–23]. Similarly, RT 078 has only been reported in South Korea and China [24,25]. The ribotypes most commonly reported in Asia are RT 017, 018, 014, 002 and 001 [24–29]. These ribotypes are among the top ten most commonly found ribotypes in Europe [30]. Of note, RT 017, which is a toxin A-negative, toxin B-positive (A− B+ ) strain, is the predominant strain in China and South Korea [24,25,31] and is prevalent in Japan, Taiwan and Hong Kong [29,32–35]. This ribotype has also caused epidemics in The Netherlands and Ireland [36,37] and is an emerging ribotype in Australia [38]. Only one study investigating C. difficile in production animals in Asia has been published, reporting a low prevalence of 0.8% (2/250) among finishing pigs aged 13–27 weeks in Japan. One isolate was a toxin A− B+ strain, whilst another was PCR-negative for both toxin genes. Analysis of the toxigenic isolate revealed partial DNA sequence similarity to RT 017 [39]. 4. Study of C. difficile in Thailand A systematic search of PubMed yielded 15 papers mentioning C. difficile and Thailand [40–54]. Three additional articles of a similar nature were obtained through a manual search of various Thai

databases [55–57]. Of the 18 studies mentioned, 9 described the prevalence of C. difficile-associated diarrhoea (CDAD), 6 evaluated C. difficile detection methods, 4 described clinical features of C. difficile-associated disease and 2 investigated the susceptibility of C. difficile to antimicrobials. Only two studies reported molecular typing results using pulsed-field gel electrophoresis (PFGE), and no data on circulating PCR ribotypes have been published. In addition, there has been no work published on C. difficile in animals or the environment in Thailand. 4.1. Prevalence of C. difficile The first prevalence study was conducted in 1990 among inpatients and outpatients of all ages at Siriraj, Ramathibodi and Children’s Hospitals in Bangkok. The study reported an overall faecal cytotoxin prevalence of 52.2% (106/203) in diarrhoeal stools compared with 22.4% (17/76) in the control group [53]. By culture, C. difficile was recovered from only 4.8% (13/269) of the diarrhoeal group and 2.6% (3/114) of the controls. In infants aged 0–3 years old, antimicrobial exposure appeared to be a risk factor for CDAD. Faecal cytotoxin was detected at a higher frequency in antimicrobialtreated (60.9%) compared with non-antimicrobial-treated infants (50.5%) [53]. Following this first publication, a study was conducted to assess the incidence of CDAD among adult patients (>15 years old) at Siriraj Hospital between 1991 and 1994 [46]. This study compared patients who received either clindamycin or a ␤-lactam antimicrobial with non-antimicrobial-treated controls. Most patients who received ␤-lactams were treated with ampicillin or cephalosporins (cefazolin, cefotaxime, ceftriaxone and ceftazidime). No patient had a history of diarrhoea, exposure to antimicrobials within 30 days prior to enrolment, underlying gastrointestinal illness, granulocytopenia or diabetes mellitus. The incidence of diarrhoea was not significantly different between the three groups, however toxin A was detected significantly more frequently (P = 0.004) among clindamycin-treated (10.7%; 15/140) and ␤-lactam-treated patients (10.0%; 14/140) than among the controls (1.4%; 2/140). The overall prevalence of CDAD was also significantly higher in the antimicrobial-treated groups (14.3%; 20/140) than in the controls (0.7%; 1/140) (P = 0.02) [46]. The discrepancy between the prevalence of CDAD in the previous study (52.2%) compared with this study may be due to differences in detection method used [tissue culture cytotoxin assay in the first publication and toxin A enzyme immunoassay (EIA) (TechLab, BioWhittaker) in the second] or to differences in patient characteristics (84.8% of patients in the first publication were aged between 0 and 3 years, whilst the second publication only included >15 years old). However, the fact that only 4.8% of the diarrhoeal group and 2.6% of controls were culture-positive [53] suggests technical problems in either the faecal cytotoxin test or the culture method. Three studies published between 1998 and 2001 investigated the prevalence of C. difficile among immunocompromised patients [48,52,55]. The first of these detected faecal toxin A (TechLab, Bio Whittaker) in 36.7% (11/30) of febrile neutropenic paediatric oncology patients at Siriraj Hospital [55]. None of the patients in the study had abdominal cramps or diarrhoea, suggesting that asymptomatic colonisation was more likely. The second study investigated C. difficile prevalence in a human immunodeficiency virus (HIV)-positive cohort [52]. Significantly more faecal toxin (Meridian PremierTM ) was detected in HIV-positive diarrhoeal patients (58.8%; 20/34) than in HIV-negative diarrhoeal patients (36.5%; 99/271) (P = 0.012). Toxin A was detected in 12.6% (21/167) of HIV-positive non-diarrhoeal patients. The study also reported six different PFGE types among 11 isolates, which were designated with internal nomenclature (types A–F). The third study investigated the prevalence of enteric pathogens in diarrhoeal patients

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with acquired immunodeficiency syndrome (AIDS) at Siriraj and Bamrasnaradura Hospitals. C. difficile toxin A was detected by EIA (OxoidTM ) in 15.6% (16/102) of HIV-positive patients [48]. In 2001, Wongwanich et al. investigated the prevalence of C. difficile among asymptomatic children as well as adults with antimicrobial-associated and non-antimicrobial-associated diarrhoea at Siriraj Hospital. Faecal specimens were cultured onto cycloserine–cefoxitin–fructose agar and the presence of the tcdA gene was confirmed with an in-house PCR [51]. C. difficile was isolated from 11.9% (28/235) and 21.1% (16/76) of asymptomatic infants (60-year-old age group, although the difference compared with other age groups was not significant (P = 0.287). The study reported 14 PFGE types and 8 subtypes among 77 C. difficile strains isolated [51]. In 2003, researchers investigated the prevalence of C. difficile among 284 patients admitted between January 2000 and May 2001 for suspected antimicrobial-associated diarrhoea at Siriraj Hospital. An additional 290 faecal specimens obtained from government and private hospitals in Thailand were also included [54]. C. difficile was isolated from 18.6% of patients (107/574). Of the 107 culturepositive stools, 44.9% were tcdA- and tcdB-positive by PCR and 46.7% were toxin A/B-positive by an EIA (Meridian Premier CytocloneTM ). Concordance between the two methods was observed in 97 stool samples (90.6%). A similar albeit slightly higher prevalence of toxin genes was reported when PCR was performed on the C. difficile isolates (48.6%). Only one isolate was positive for tcdB but negative for tcdA. The study also found that 13.1% of non-toxigenic isolates (14/107) had a positive stool tcdA and tcdB result, suggesting possible co-infection with multiple strains of C. difficile [54]. In 2011, a retrospective study on inpatients (>14 years old) at Siriraj Hospital from January–December 2008 was published [47]. The aim was to determine the prevalence, risk factors, treatments and outcomes of nosocomially acquired CDAD. C. difficile toxins A and B were detected in 12.3% (25/203) of diarrhoeal patients by an immunochromatographic method (Remel Xpect® ). Exposure to antineoplastic drugs, multiple antimicrobials or proton pump inhibitors, and haematological malignancy were independent risk factors associated with development of CDAD. Most patients were diagnosed by the detection of toxin in stool and seven by the presence of PMC on colonoscopy. Metronidazole was used to treat 82.3% of the cases (74.5% response rate), whilst vancomycin was used only in severe cases. Mortality due to CDAD was 3.2% (2/62) [47]. The most recent study was published in 2012. The aim was to determine the prevalence of hospital-acquired CDAD in patients (≥15 years old) at Ramathibodi Hospital [41]. Of 175 stool specimens, 26.9% of patients were positive for C. difficile. These included 12.6% who were positive for toxins A/B by EIA (VIDAS® ; bioMérieux) and 24% who were positive for tcdB by PCR. Concordance between the two methods was 83.2%. Leukocyte counts of >15,000 cell/mm3 and development of diarrhoea after ≥10 days of antimicrobial administration were significantly associated with CDAD. Mortality due to C. difficile was reported to be 6.4% [41].

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In summary, the prevalence of CDAD in Thailand ranged from 4.8% to 52.2%, depending on detection methods, patient demography and exclusion criteria. Variations in these factors make comparison of the data from various studies difficult. It was noteworthy that most prevalence studies were conducted among patients admitted at Siriraj Hospital, the biggest hospital in Thailand with 2300 beds. Although this is not unexpected, given the prominent role of the institution in providing healthcare services in Thailand, further studies should be conducted at other healthcare facilities in order to obtain a more complete overview of the prevalence of C. difficile in this country. 4.2. Evaluation of C. difficile detection methods used in Thailand In the early 1990s, the National Institute of Health (NIH) of Thailand published two studies evaluating a latex agglutination test for detection of C. difficile toxin A (CD D-1) [43,49]. The findings suggested that the assay was not specific for toxigenic C. difficile. This was later confirmed by numerous studies which showed that the kit did not detect toxin A but rather the glutamate dehydrogenase (GDH) enzyme produced by all C. difficile and some other species of bacteria [49,58,59]. A feature of many early studies in Thailand was the use of a toxin A EIA as the sole method of C. difficile detection [42,45,46,48,55]. Given the high prevalence of RT 017 (a toxin A-negative strain) in this region [26] and the low sensitivity of EIA, widespread use of toxin A EIA alone is likely to have led to an underestimation of the true prevalence. In 2003, a study comparing the performance of toxin A/B EIA (Meridian Premier CytocloneTM ) and PCR for tcdA and tcdB in detection of C. difficile in culture-positive stool specimens reported a good correlation between the two techniques [54]. Discordant results occurred in only 9.3% of the samples (10/107) and may have been due to the degradation of toxins during transportation or to cross-reactions with C. sordellii toxin. In addition, 16.8% of toxigenic strains (18/107) were isolated from PCR-negative specimens, possibly due to the presence of PCR inhibitors in the sample [54]. Such observations emphasise the importance of an appropriate and timely way to transport specimens in resource-poor settings. A similar study published in 2012 showed PCR for tcdB to be twofold more sensitive than a toxin A/B EIA (VIDAS® ; bioMérieux) for detecting C. difficile in stool specimens [41]. The discrepancies, however, may have been accounted for by the use of the least sensitive commercially available EIA in this study [60]. The most recent study undertaken in Thailand [40] had three main aims: (i) to develop and assess the sensitivity and specificity of an in-house multiplex PCR for the detection of tcdA, tcdB, binary toxin genes (cdtA/B) and the 16S rDNA gene; (ii) to optimise sample processing by enhancing spore germination; and (iii) to test the efficacy of the optimised techniques in the detection of C. difficile in clinical specimens. When tested with multiple bacterial species, the test appeared specific for C. difficile. It was also sensitive, with a detection limit of 22 genomic copy numbers per reaction. The optimal incubation condition was non-selective enrichment at 37 ◦ C for 1 h in brain–heart infusion broth supplemented with sodium taurocholate. This had a detection limit of 5 spores/g of faeces. The multiplex PCR was further compared against toxigenic culture and toxin A/B EIA (VIDAS® ; bioMérieux) using 238 faecal samples collected from patients with suspected CDI. All samples were positive for the 16S rDNA gene. Agreement between the three methods was observed for 76.5% (182/238) of the samples. Using toxigenic culture as the gold standard, concordance was observed with PCR and EIA in 88.2% (210/238) and 86.6% (206/238) of the samples, respectively [40]. Although the multiplex PCR exhibited high sensitivity and specificity for tcdA and tcdB, the gel electrophoresis image of various C. difficile PCR ribotypes suggested an issue with primer design. The image shows multiple known binary

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toxin-negative PCR ribotypes to have positive binary toxin gene PCR results (including RT 001, 020, 029, 046, 056, 070, 077, 095 and 106). 4.3. Antimicrobial susceptibility of C. difficile Researchers from NIH Thailand published two studies in 1994 and 1996 investigating the antimicrobial susceptibility of C. difficile strains isolated from Thai patients. The aim of the first study was to assess the susceptibility of 28 C. difficile strains and 11 non-C. difficile clostridia obtained from colitis and diarrhoeal patients to 16 antimicrobials [56]. The identity of presumptive C. difficile isolates was confirmed by positive leucine arylamidase activity and the presence of GDH enzyme by a latex agglutination test (CD D-1; Mitsubishi Chemical Inc.). Using a broth-disc elusion technique, all isolates were susceptible to carbenicillin, metronidazole and vancomycin. Among other antimicrobials, piperacillin was most active (4% resistance), followed by rifampicin, chloramphenicol, ticarcillin and penicillin, which showed intermediate activities (14–32% resistance). A high degree of resistance (≥50%) was observed for cefazolin, cefoperazone, tetracycline, erythromycin, clindamycin, ampicillin, bacitracin and cefoxitin, the latter being the least active. Twenty-seven antimicrobial resistance profiles were observed. All but one isolate of C. difficile were resistant to two or more antimicrobials. The second study evaluated the activity of four antimicrobials against 38 strains of C. difficile isolated from patients with colitis and diarrhoea [50]. Using Etest (AB BIODISK), the MIC90 values (minimum inhibitory concentrations that inhibit 90% of the isolates) of teicoplanin, vancomycin, metronidazole and clindamycin were 0.5, 2.0, 0.5 and ≥256 ␮g/mL, respectively. According to the Clinical and Laboratory Standards Institute (CLSI) MIC breakpoints for each antimicrobial, all isolates were susceptible to teicoplanin, vancomycin and metronidazole. Resistance to clindamycin was observed in 39.5% of isolates. Teicoplanin appeared to be approximately fourfold more potent than vancomycin, indicating its potential as a therapeutic drug option. Consistent with the literature, there was no resistance to metronidazole or vancomycin, which are currently recommended for treatment of CDI [61]. 4.4. Clinical features, treatment and outcomes associated with C. difficile infection in Thailand From July 1993 to August 1994, 34 oncology patients at Chulalongkorn Hospital developed diarrhoea and colitis following administration of antineoplastic agents [45]. Diarrhoeal samples from six patients tested positive for C. difficile toxin A by EIA (Meridian PremierTM ). Clinical records demonstrated gastrointestinal malignancy to be the underlying illness in three patients, and five were treated with 5-fluorouracil (5-FU). No patient had received antimicrobials in the 6 weeks leading up to the onset of diarrhoea. All patients developed moderate-to-severe diarrhoea requiring hospitalisation and fluid replacement, with one patient developing high fever (38.5 ◦ C). Diarrhoea resolved after vancomycin administration in three patients and without specific treatment in others. All diarrhoeal episodes resolved within 4–10 days. The study demonstrated that 5-FU is a potential risk factor for CDI and alerts physicians to consider C. difficile as an aetiological agent of diarrhoea in oncology patients who have been exposed to antineoplastic agents, with or without recent exposure to antimicrobials [45]. Later, a retrospective study was conducted to investigate risk factors, clinical features, treatment and outcomes of patients with CDAD at the same hospital [42]. All patients whose diarrhoeal samples tested positive for C. difficile toxin A by EIA (OxoidTM ) between January 2002 and July 2005 were included. Of the 88

potential subjects, 56 patients aged 4 months to 93 years had accessible medical records and were included in the analysis. Of these, 89.3% had an underlying disorder, with malignancies being the most common (51.8%). All patients had been exposed to at least one antimicrobial in the preceding 60 days, with cephalosporins and carbapenems being most common, and 25 (44.6%) received gastric suppressants (omeprazole or ranitidine) that were administered between 5 days and 170 days prior to the onset of diarrhoea. Chemotherapeutic agents were used in 12 patients (21.4%) and were administered between 5 days and 48 days prior to the diagnosis. Eight patients (14.3%) did not receive specific treatment and only one of these showed clinical improvement. Among those who received treatment, 38 (67.9%), 4 (7.1%) and 2 (3.6%) received oral metronidazole, intravenous metronidazole and oral vancomycin, respectively. Of 48 treated subjects, 32 (66.7%) showed clinical improvement (diarrhoea resolution) within 11 days, 12 (25.0%) did not show improvement and 2 (4.2%) had recurrent CDAD following treatment with oral metronidazole. Of the 12 patients who did not respond to the treatment, 4 did not discontinue the agent suspected to cause CDAD. The all-cause mortality was 37.5% (21/56), with none being a C. difficile-related death [42]. With oral metronidazole as the main therapeutic drug, the response rate of 66.7% was lower than the rate observed in studies published prior to 2002 [90% (Canada) to 98% (USA)] [62,63] but similar to more recent data [74.3% (Canada) to 74.5% (Thailand) for metronidazole] [47,63]. The most recent study was published in 2013 with the aim of describing the computed tomography (CT) features of patients with clinically proven C. difficile colitis [44]. All patients hospitalised at Siriraj Hospital between January 2006 and June 2009 who had either a positive faecal toxin A/B by immunochromatographic assay (Remel Xpect® ) or endoscopically proven colitis were included. Fifteen patients satisfied the criteria and had an appropriate CT scan within 4 weeks of stool collection. Pancolonic wall thickening, mild pericolonic stranding and accordion sign appeared to be the key features of C. difficile-associated colitis [44]. CT is an attractive option for patients with suspected C. difficile-associated colitis who, due to the underlying conditions, cannot undergo colonoscopy. Despite the small sample size, this study provided valuable information to assist physicians in clinical diagnosis. In summary, the risk factors, clinical features, treatment and observed outcomes were similar to those reported in the existing literature from developed countries.

5. Antimicrobial usage in Thai livestock and patients As with humans, antimicrobial exposure is the major risk factor for CDI in animals [64]. In Thailand, there appears to be widespread use of antimicrobials both in the livestock and aquaculture sectors. Animal drugs manufactured in Thailand rose almost four-fold between 1996 and 2005 [65]. Reports from the Thai Drug Control Division indicated that tetracyclines, macrolides, trimethoprim, aminoglycosides and penicillins were the most used drugs in food animals [66]. Despite efforts by the Department of Livestock Development and Ministry of Agriculture and Cooperatives to control and monitor antibiotic usage, inappropriate use of antibiotics by smallto medium-scale farmers remains common [67]. This is partly due to the limited financial resources available to farmers, which leads them to accessing cheap and often inappropriate antimicrobials. Owing to under-reporting of use, data regarding antimicrobial usage may be an under-representation of the true prevalence [67]. In 2007, a study was conducted among 101 broiler farms (700–18,000 broilers per farm) in Songkhla, Thailand, with the aim of documenting the pattern and determinants of antimicrobial usage [66]. Researchers found that antimicrobials were used by all farms for prophylactic purposes. The drugs most commonly

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used were enrofloxacin, amoxicillin, doxycycline, colistin and roxithromycin. The combination of amoxicillin and enrofloxacin was the most frequently used. Ninety-five percent of small-scale farms were controlled by a few large enterprises, and their choice of antimicrobial depended on their employer or the contracting body [66]. In dairy farms, cloxacillin appeared to be the most used antimicrobial, comprising >80% of the intramammary drugs being sold in Northern Thailand. Other antimicrobials used to treat gastrointestinal illnesses in cattle include trimethoprim/sulfamethoxazole, tetracycline and gentamicin [65]. Antimicrobials were also used extensively in the aquaculture industry. A survey to determine the use of chemical and biological products in 76 shrimp farms from three major farming areas in Thailand [68] reported the use of at least one antimicrobial by 74% of the farmers. The drugs were often incorporated into the feed and were used for prophylactic and therapeutic (e.g. against Vibrio infection) purposes. The classes of antimicrobial most frequently used included fluoroquinolones (e.g. norfloxacin, enrofloxacin), tetracyclines (e.g. oxytetracycline) and sulfonamides. Use of chloramphenicol in animal feed is prohibited in Thailand. As there is no legislation on the use of antimicrobials in aquaculture, a Code of Conduct containing recommendations and guidelines for shrimp farming was developed by the Department of Fishery and Thai Marine Farmers Association. Very few farmers included in the study knew of its existence [68]. These findings strongly suggest that more educational programmes must be carried out, together with the implementation of such guidelines, in order for any changes to be realised. This issue is not limited to animals but also occurs for human antimicrobial use. In 1990, a study conducted in Siriraj Hospital to evaluate the level of irrational antimicrobial usage showed that only 8.8% of prescriptions (27/307) were appropriate, with the main reason for misuse being use without evidence of infection (35.8%; 110/307) [69]. Antimicrobials were routinely administered before test results became available and as prophylaxis in those without symptoms [69]. These included cephalosporins (11%; 71/655), which have relatively high attributable risk for CDAD [70]. In the community, pharmacists are authorised to sell most antimicrobials (intended for human use) without the need for a prescription. This practice creates settings where misuse is the likely outcome, particularly in rural areas where exposure to livestock is more common. Collective efforts both from the public and healthcare sectors are essential in reducing indiscriminate and inappropriate antimicrobial use, which will assist in lowering the spread of antimicrobial-resistant bacteria [65].

6. Thai swine industry in relation to C. difficile Prior to 1960, the swine industry in Thailand consisted mainly of small-scale farmers. The four main native breeds, often raised in backyards, include Kwai, Raad, Phung and Hainan [71]. Although native breeds are more resilient and well adapted to the hot and humid climate in Thailand, they grow slowly (ca. 180–350 g/day), have a low reproductive rate (ca. 6–8 pigs/litter) and produce low-protein and high-fat content meat [71,72]. In the 1960s, the Department of Livestock Development imported exotic breeds from the UK (Large Whites, Tamworth and Berkshire) and the USA (Landrace and Duroc Jersey). These breeds were cross-bred with the native pigs to produce offspring with desirable qualities [73]. Since the 1980s, armed with a genetically improved breed, intensive swine operations in Thailand (>1000 sows and/or finishing pigs) have increased in number [71]. C. difficile is an important enteric pathogen of pigs, causing diarrhoea in piglets [30]. The PCR ribotype most commonly found among pigs in North America and Europe is RT 078 [11,74,75]. In

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Australia, RT 078 has not been documented among pig herds [76]. Since pigs in commercial farms in Thailand originally came from North America or Europe [77], C. difficile distribution in commercial herds may be similar to that of the origin of the animals. Integrated livestock–fish farming systems are commonly used throughout Southeast Asia, including Thailand. The main feature of these systems is the use of manure from livestock, including pigs, chickens and ducks, as fish feed [78]. Such practices have implications for the transmission of animal pathogens, including C. difficile, which is transmitted faeco-orally. Although there is a clear trend towards intensive farming [79], backyard-style free-range farming systems and the livestock–fish integrated farming systems are still prevalent in many parts of Thailand [80,81]. Animals, including pigs and poultry, can act as a reservoir of infection for C. difficile in the community and further study is warranted.

7. Conclusions As described, there is very little information regarding the epidemiology of C. difficile in humans, animals and the environment in Thailand. Given the high level of indiscriminate and inappropriate use of antimicrobials both in humans and animals in this region, it is highly probable that CDI is relatively common. Despite this, the level of awareness among Thai physicians is extremely low, and current diagnostic procedures and typing methods are not optimum. Even lower levels of awareness exist among veterinarians and farmers. As such, it is essential that more surveillance of C. difficile is done both in hospital and community settings to elucidate its epidemiology. More study should also be done to investigate the molecular characteristics and antimicrobial susceptibility profiles of Thai C. difficile isolates. This information will not only assist physicians in patient management and infection control, it may also improve livestock quality and hence the economic fortunes of farmers. Funding: Scholarship for International Research Fee awarded by the University of Western Australia to P.P.; Chalermphrakiat Grant to P.K. Competing interests: None declared. Ethical approval: Not required.

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