Vol. 10 No. 1 January–April 2022 Available online at IJTID Website: https://e-journal.unair.ac.id/IJTID/ Review Article Clostridium diffi cile Infection (CDI) by Hypervirulent BI/NAP1/027 Strain: a Comprehensive Review of Toxigenicity, Pathogenesis, Risk Factors, and Preventative Measures Ni Nyoman Sri Budayanti1, I Gusti Putu Suka Aryana2*, Ni Luh Putu Harta Wedari3 1Clinical Microbiology Department, Faculty of Medicine, Universitas Udayana, Universitas Udayana Hospital, Bali, Indonesia 2Geriatric Division, Internal Medicine Department, Faculty of Medicine, Universitas Udayana, Sanglah General Hospital, Bali, Indonesia 3Clinical Microbiology Specialist Program, Faculty of Medicine, Universitas Udayana, Sanglah General Hospital, Bali, Indonesia Received: 29th October 2021; Revised: 17th January 2022; Accepted: 26th January 2022 ABSTRACT Clostridium diffi cile is an anaerobic bacil gram-positive bacteria, able to form spores and toxin, that is transmitted among humans through the fecal–oral route. Clostridium diffi cile infection (CDI), a typical nosocomial infection has been contributed to a signifi cant proportion of morbidity and mortality among in-patients with a case-fatality rate of 14% within 30 days after diagnosis. Profound culture and toxin examination for C. diffi cile are still minimal in many hospitals in various Asian countries. Consequently, C. diffi cile reports in Asia remain rare. Highly virulent form of C. diffi cile caused greater fatality and epidemics severity. Elderly age, hospitalization, exposure to antibiotics e.g., cephalosporins, fl uoroquinolones, clindamycin, and penicillin contributed as main risk factors. Hypervirulent strain BI/NAP1/027 demonstrated to carry CdtLoc gene locus encodes CD196 ADP-ribosyltransferase (CDT) or known as binary toxin. Virulence factors are TcdA, TcdB, CDTa CDTb in which hypersporulation and mutation of TcD gene by hypervirulent strain led to toxin hyperexpression. Early cases detection, building management team to evaluate patient positive with all C. diffi cile toxins, hand hygiene improvement, continuation of contact precautions after diarrhea resolution, audit of infection control, and restriction of antimicrobials should be implemented as preventative measures. Focus measures also should emphasize on development of vaccine of C. diffi cile to boost immune state of elderly people. This review aims to describe severity of disease caused by hypervirulent BI/NAP1/027 C. diffi cile strain, its mechanism or pathogenesis, risk factors, current treatment options available, along with proposed preventative measures and infection control. Keywords: Clostridium diffi cile infection (CDI), hypervirulent strain, BI/NAP1/027 ABSTRAK Clostridium diffi cile adalah bakteri basil gram positif anaerobik, pembentuk spora dan toksin, yang ditularkan di antara manusia melalui rute fekal-oral. Clostridium diffi cile infection (CDI), sebuah tipikal infeksi nosokomial telah berkontribusi pada proporsi yang signifi kan terhadap morbiditas dan mortalitas di antara pasien rawat inap dengan tingkat fatalitas kasus 14% dalam waktu 30 hari setelah diagnosis. Kultur dan pemeriksaan toksin C. diffi cile masih minim di banyak rumah sakit di berbagai negara Asia. Akibatnya, laporan C. diffi cile di Asia masih jarang. Epidemi kematian dan keparahan yang lebih besar dari CDI disebabkan oleh C. diffi cile yang hipervirulen. Faktor risiko utama adalah usia lanjut, rawat inap, paparan antibiotik misalnya sefalosporin, fl uoroquinolones, klindamisin, dan penisilin. Strain hipervirulen BI/NAP1/027 terbukti membawa lokus gen CdtLoc yang mengkode CD196 ADP-ribosyltransferase (CDT) atau dikenal sebagai toksin biner. Faktor virulensi yaitu TcdA, TcdB, CDTa CDTb; strain hipervirulen mampu melakukan hipersporulasi dan mutasi gen TcD yang menyebabkan hiperekspresi toksin. Tindakan pencegahan dapat dilakukan dengan deteksi dini kasus, pembentukan tim manajemen untuk mengevaluasi pasien yang positif semua toksin C. diffi cile, peningkatan kebersihan tangan, kelanjutan tindakan pencegahan kontak setelah resolusi diare, audit pengendalian infeksi, dan pembatasan antimikroba. Fokus upaya juga sebaiknya ditekankan pada pengembangan vaksin C. diffi cile untuk meningkatkan * Corresponding Author: ptsuka_aryana@unud.ac.id IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA Share alike 4.0 28 Indonesian Journal of Tropical and Infectious Disease, Vol. 10 No. 1 January–April 2022: 27–41 status kekebalan pada individu berusia lanjut. Tinjauan ini bertujuan untuk menggambarkan tingkat keparahan penyakit yang disebabkan oleh strain C. diffi cile BI/NAP1/027 hipervirulen, mekanisme atau patogenesisnya, faktor risiko, pilihan pengobatan yang tersedia, serta tindakan pencegahan dan pengendalian infeksi. Kata kunci: Clostridium diffi cile infection (CDI), strain hipervirulen, BI/NAP1/027 How to cite: Budayanti, N. N. S., Aryana, I. G. P. S., Wedari, N. L. P. H., Clostridium diffi cile Infection (CDI) by Hypervirulent BI/NAP1/027 Strain: a Comprehensive Review of Toxigenicity, Pathogenesis, Risk Factors, and Preventative Measures. Indonesian Journal of Tropical and Infectious Disease, 10(1), p. 27–41, Apr. 2022. INTRODUCTION Clostridium difficile infection (CDI) has been known as a typical nosocomial infection and contributes to a signifi cant proportion of morbidity and mortality among in-patients with a case-fatality rate of 14% within 30 days after diagnosis.1 C. diffi cile gives rise to numerous infections varying from mild diarrhea to pseudomembranous colitis (PMC), mainly in elderly patients with antibiotic treatment. In addition, high healthcare costs related to CDI increase the fi nancial burden of government on health expenditure. It was recorded that half a million infections were attributed to CDI in the United States in 2011 with an incidence rate of 8.75 cases/1,000 adult admissions in 2009.2,3 A literature study by Collins et al.4 found few data of CDI cases. The study found that a study in Japan only reported on the ribotyping result of C. diffi cile without any information on CDI prevalence or incidence in Japan; CDI incidence increased from 1.7/1,000 to 2.7/1,000 adults in Korea, and 17.1/10,000 inpatients in Shanghai were attributed to CDI. Meanwhile, about 44% and 14% of colitis positive patients were positively diagnosed with the C. diffi cile toxin in Philippine and Malaysia, respectively.4 A more recent study showed that CDI prevalence was 9.2% in Thailand.5 There are only a few reports about CDI incidence or prevalence in Indonesia. A study reported that there were eight types of C. diffi cile strains presenting in healthy people,6 while another study showed that the prevalence of C. diffi cile (toxin A) was 1.3% in community and hospital in Jakarta.7 The last report originated from Central Java showing the prevalence of CDI to be 20.6% by 2017.8 Profoundly extensive culture and toxin examination for C.diffi cile are still minimal in many hospitals in various Asian countries. Consequently, C.difficile reports in Asia remain rare. In the current study held in Malaysia, assays determining toxin A/B from 175 stool samples collected from patients with antibiotic-associated diarrhea have been performed in tertiary hospital in north-eastern suburb; 24 of them (13.7%) tested positive for toxin, where the age most of infected patients is >50 years.9-11 However, no ribotyping or any other molecular test have been done in regard to isolates of Malaysian C. diffi cile. Similar to Malaysia, CDI cases reporting in Indonesia is uncommon. It has been found 1.3% test results of stool sample reveal the etiology of diarrhea in Indonesia children was C. diffi cile. Furthermore, only enzyme immunoassay of toxin A was conducted; therefore, the C. diffi cile true prevalence may have been substantially greater. Molecular study of eight isolates collected from Indonesia established fi ve of the results identifi ed as toxinotype VIII and ribotype 017, assembled into epidemic strains of international 017. Two of them are A+B+ toxinotype 0, and one remaining A-B+ isolate was identifi ed as toxinotype XVI binary toxin.12-14 Some risk factors including advanced age, antibiotic exposure, and hospitalization are highly associated with CDI.15 Regulation of antibiotic usage in Asian countries is considered to be poor. There has been a review in Southeast Asian countries which depicted 47% of pneumonia cases as not receiving proper antibiotic whereas 54% of patients with diarrhea were receiving antibiotic unnecessarily, with 40% of under- dose antibiotics prescribed. The advanced age individuals with recent antibiotic treatment are at IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA Share alike 4.0 29Ni Nyoman Sri Budayanti, et al.: Clostridium difficile Infection (CDI) by Hypervirulent the highest risk for CDI as they lack of benefi cial gut microbiota and have low immunity due to age and other comorbidities.16-20 This group is excessively aff ected and has the highest mortality rate due to CDI with 2% of risk increase every year after 18 years old of age. A report described around one in ten deaths due to CDI in advanced age people in the USA in 2010. There were no data of CDI on advanced age people in Indonesia, which could be due to lack of surveillance on CDI cases followed by limited laboratory facility in the hospital capable of diagnosing CDI. Besides, recurrence (relapse/reinfection) and death cases due to CDI in advanced age people would be higher because of improper treatment.21 Severe form of C. diffi cile infection (CDI) is caused by hypervirulent strain identifi ed as type 1 of North American pulsed fi eld, type B1 class from analysis of restriction-endonuclease, ribotype 027 as presented by PCR. Hypervirulent strain leads nationwide CDI outbreaks in European countries, Canada, and the United States (U.S.). First outbreak report of type 027 CDI was in Canada where the worst infected was Quebec in 2005. In the U.S., type 027 CDI aff ected 38 states. Meanwhile based on European Centre for Disease Prevention and Control, there were infections in 16 countries due to type 027 CDI. Hypervirulent strain of toxinotype III nurture TcdA and TcdB toxin genes, possess deletion of 18-bp in TcdC of toxin regulatory gene, and deletion at area 117. It leads to premature stop codon and frameshift, causing TcdC protein truncation. The rising case of type 027 virulence associated with more excessive toxin production can be attributed to lack control of regulatory TcdC.19-21A cohort study estimated that around 40% of CDI cases were community-acquired CDI (CA-CDI). CA- CDI occurs in younger age people, less severe symptoms, shorter hospital stay, lower recurrence rate and no deaths have been reported attributable to CA-CDI. Besides, CDI is also exacerbated by the discovery of hypervirulent strains and antibiotics resistant to quinolones, gatifl oxacin instead of levofl oxacin.17-19 The emerging of CA-CDI will be a risk factor for domestic and foreign tourists who visit Bali. Since 2000, greater fatality and severity epidemics of CDI have been caused by a highly virulent form of C. diffi cile. BI/NAP1/027 strains have spread widely and robustly over past 10 years and have been associated to CDI epidemics. The prevalent ribotypes in the Middle East are 140, 126, 078, 046, 014, 002, and 001, meanwhile the more prevalent ribotypes in Asia are 018, 017, 014, 002, and 001. In North America and Europe, ribotypes 078, 027, 020, 014, and 001 have been the uppermost strains.22-24 Ribotype 027 has been found to possess reduced susceptibility to chloramphenicol, imipenem, clindamycin, moxifl oxacin, rifampicin, and metronidazole. These characteristics implicate to more severe presentation of disease, high morbidity and mortality rates due to antimicrobial resistance juxtaposed to other strains. Spores of ribotype 027 expand more robustly and easily in hospital as they able to resist disinfectants, cleaning, and hospital surroundings. Observational study on patients with diarrhea in Veteran Aff airs Medical Center, U.S. demonstrated around 22% of them were positive of BI/NAP1/027 strain.19-24 This literature review aims to describe severity of disease caused by hypervirulent BI/NAP1/027 C. diffi cile strain, its mechanism or pathogenesis, risk factors, current treatment options available, along with proposed preventative measures and infection control.22-24 Clostridium diffi cile INFECTION (CDI) Clostridium diffi cile is an anaerobic gram- positive bacillus bacterium, able to form spore and toxin, transmitted in humans by fecal–oral pathway. In the U.S., C. diffi cile is the most frequent nosocomial pathogen reported. A surveillance study of 2011 found 453,000 CDI cases with 29,000 associated deaths, wherein around a quarter of those were community- acquired. Nosocomial C. difficile infection quadruples hospitalizations cost causing rise of expenditures by about $1.5 billion in the U.S. yearly. It was recorded that half a million infections were attributed to CDI in the United States in 2011 with an incidence rate of 8.75 cases/1,000 adult admissions in 2009. In Hong IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA Share alike 4.0 30 Indonesian Journal of Tropical and Infectious Disease, Vol. 10 No. 1 January–April 2022: 27–41 Kong, there were more than fi fteen thousand CDI cases from 2006 to 2014 in which most were identifi ed as a nosocomial infection. A nationwide study in Korea revealed CDI total incidence was 2.7 cases/1,000 adult admissions in 2008. CDI is also known for its propensity to recurrence among 35% of patients with antibiotic therapy and more than a half of recurrences of CDI are identifi ed as relapse (relapse or reinfection).25 Due to CDI, approximately $1.1 billion are utilized in healthcare cost annually in the USA, while about €3 million is associated with healthcare costs in Europe. Compared to reports from countries across Europe and the USA, the prevalence of CDI in Asia is not fully known. In Korea, survey across 17 tertiary hospitals, from 2004 until 2008, found CDI incidence cases soared from 1.7/1,000 to 2.7/1,000 adults. Community-acquired CDI (CA-CDI) proportion over total CDI cases in a hospital in Busan was 7.1%, meanwhile 59.4% of CDI cases at a Seoul hospital’s emergency department were CA-CDI. Based on a comprehensive study in Shanghai, China from March 2007 until April 2008, overall CDI incidence was 17.1/10,000 admissions; mild CDI because of younger mean age (62.8 years) compared to 63% patients were ≥65 years in a comprehensive European study. In addition, a survey across 13 Asia-Pacifi c countries demonstrated the proportion of CDI associated to healthcare facility was 53.6% and CA-CDI was 16.5%. CDI case reports in Indonesia remain uncommon. C. diffi cile was identifi ed in 1.3% stool samples of Indonesian children. However these data were not enough to reflect global prevalence in Asia. Furthermore CDI prevalence data of elderly are still unavailable to date.22-26 CDI mostly occurs in advanced age people, which is possibly explained by some of the risk factors, including frequent exposure to healthcare, age-related changes in physiology, increasing antibiotics usage, changes in the composition of gut fl ora, and increased comorbidities. Frequent exposure to healthcare increases the opportunity of contacting with environments contaminated with endospores of C. diffi cile and frequent utilization of antimicrobials. Carriers of C. diffi cile, both asymptomatic and symptomatic, could contain spores on their skin and discard those into the environment. Age-related physiologic changes also increase the risk of CDI, particularly changes in the immune system. The development and recurrence of CDI have been associated with the ability to generate immune responses, and the ability to produce antibodies against toxin may aff ect the progress of colonization and active infection. Aging is accompanied by immune senescence – a degeneration of the immune system related to advanced age – and it has been associated with a diminishing adaptive immune system.27-29 C. diffi cile has the ability to do colonization in large intestine, then releasing exotoxins protein (TcdA, TcdB) leading to colitis in people with risk factors. Figure 1 depicts TcdA and TcdB arbitrate C. difficile diarrhea, causing Rho family members’ inactivation, Rho GTPases (guanosine triphospatases). This is followed by neutrophilic colitis, colonocyte death, functional loss of intestinal barrier, and death of colonocytes. Expression of clinical CDI disease is exerted by host immune responses and strain of C. diffi cile. A dramatic increase of severe CDI in hospitals was initially reported in the beginning of 2000s. CDC (Centers for Disease Control and Prevention) depicted isolates were group BI of restriction endonuclease, NAP1 (gel electrophoresis of North American), and PCR (polymerase chain reaction) 027; therefore, as BI/NAP1/027. This strain’s characteristics are high level resistance of fl uoroquinolones, robust production of toxin, effi cient rate of sporulation, and signifi cantly high mortality compared to less virulent C. diffi cile.28,29 BI/NAP1/027 strain fi rstly originated in North America and Western Europe, but currently it spreads to various settings of hospitals across the globe.30,31 Even though hospital-acquired CDI has been the majority, CA-CDI has been increasing signifi cantly and contributes to a third of new CDI cases. CA-CDI happens when onset of disease begins within 12 weeks in individuals who did not stay overnight in hospitals or other healthcare facility. CA-CDI could occur in younger patients, who have unclear antibiotics IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA Share alike 4.0 31Ni Nyoman Sri Budayanti, et al.: Clostridium difficile Infection (CDI) by Hypervirulent exposure and unknown risk factors. Therefore, CA-CDI acquisition main modes are currently in investigation. CA-CDI associated morbidity and mortality remain lower compared to hospital- acquired CDI. Nonetheless, 40% of CA-CDI patients need hospitalization and relapse rates are similar to HA-CDI.32 Acid suppression infl uence to CDI remains unclear. Theoretically, gastric acid suppression allows more vegetative organisms to reach colon. However, C. diffi cile produces spores resistant to acid pH.33,34 HYPERVIRULENT BI/NAP1/027 C. diffi cile STRAIN RISK FACTORS Substantial risk factors of CDI by BI/ NAP1/027 strain are namely hospitalization, elderly age, and exposure to antibiotics, e.g., cephalosporins and fl uoroquinolones. Particular fl uoroquinolones identifi ed being risk factors are ciprofl oxacin, gatifl oxacin, moxifl oxacin, Figure 1. Progress from asymptomatic colonization to C. diffi cile infection (CDI)22-29 and levofl oxacin, presumed as consequences of fl uoroquinolones-resistance in endemic strain. Almost all antibiotics of cephalosporin class are resistant to all C. diffi cile types and have been incriminated as signifi cant risk factor in hospitals where endemic strain exists, as well as its usage for surgical prophylaxis. Consumption of agents to lower stomach acid production, e.g., proton pump inhibitors (PPI), and type 2 blockers of histamine have been recognized inconsistently as CDI risk factors in hospital with predominance of endemic strain. Besides resistance to current fl uoroquinolones, other specifi c factors of BI/ NAP1/027 strain dissemination as well as severe CDI caused by this hypervirulent strain remain speculative and need to be the substance of thorough study or research.32-34 Administration to almost all groups of antimicrobials has been delineated to cause CDI, even though cephalosporins, clindamycin, IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA Share alike 4.0 32 Indonesian Journal of Tropical and Infectious Disease, Vol. 10 No. 1 January–April 2022: 27–41 penicillin, and present fluoroquinolones are notably reported as common culprits. Numerous isolates of C. diffi cile were found containing elements of mobile genetic, markers of antibiotic resistance in chromosomes, and mutations of genetic conferring resistance into rifamycins, chloramphenicol, tetracyclines, streptogramin, lincosamide, macrolide, and fl uoroquinolones. Patients also progress to CDI disease following antibiotics therapy which leads to susceptibility of C. diffi cile strain infection. Presumably, CDI occurs due to suppression of normal microbiota in intestine for extended periods after discontinuation of antibiotics; therefore, allowing sustained opportunity for colonization and infection of C. diffi cile. Isolates resistance to clindamycin, erythromycin frequently associated to epidemics and outbreaks. Furthermore, individuals administered by clindamycin possess a remarkable high-rise CDI frequency caused by clindamycin resistant. Resistance is commonly associated with erm(B) presence encoding methyltransferase and ground in Tn5398 conjugative transposon.35 Current exploration of BI/NAP1/027 isolates has exemplifi ed re-emergence of erythromycin- resistant BI/NAP1/027 in European countries, the U.S., and Canada. Currently, therapy with fl uoroquinolones is identifi ed as BI/NAP1/027 C. diffi cile infection risk factors, and there is a proposed association between therapy with fluoroquinolones and emergence of BI/NAP1/027 strain. Even though all previous isolates of BI/NAP1/027 are susceptible to gatifl oxacin, moxifl oxacin, and fl uoroquinolones, yet resistant to levofl oxacin and ciprofl oxacin, almost all current isolates were resistant to all fl uoroquinolone antibiotics. Inhibition of DNA replication by fl uoroquinolones is as a result of its binding to DNA gyrase or topoisomerase II, or topoisomerase IV. Resistance to fl uoroquinolones in C. diffi cile is associated with particular mutations in gyrA and gyrB, that encode DNA gyrase. Fluoroquinolones are broad- spectrum antibiotics which act on gram-negative and gram-positive bacteria and are able to decrease normal fl ora in intestine, hence broad use of fl uoroquinolones in hospitals fosters spreading of BI/NAP1/027 C. diffi cile strain.32-36 H Y P E RV I R U L E N T B I / N A P 1 / 0 2 7 C . difficile STRAIN TOXIGENICITY AND PATHOGENESIS H y p e r v i r u l e n t s t r a i n B I / N A P 1 / 0 2 7 i s demonstrated to carry CdtLoc gene locus and encodes CD196 ADP-ribosyltransferase (CDT) or known as binary toxin. Hypervirulent C. diffi cile is able to produce TcdA and TcdB, similar with non-027 ribotypes throughout gene locus of PaLoc. CDT was initially isolated by Popoff et al.37 The toxin contains two distinct toxin components separately, namely CDTa and CDTb. CDTa, ADP-ribosyltransferase enzyme acts on actin modifying which leads to depolymerization and destruction of actin cytoskeleton inside gut; meanwhile CDTb hitches to gut cells and stimulates CDTa uptake. Destruction by CDT accommodates bacteria adherence and surges Toxin A and B uptake.38-40 Furthermore, hypervirulent strain contains bp frameshift deletion on TcdC gene, nucleotide 117, and functions as negative regulator of Toxin A and Toxin B. TcdC mutation causes toxins hyperexpression. Warny et al.58 demonstrated BI/NAP1/027 as able to produce 16 times of Toxin A and 23 times of Toxin B approximately compared to control strain. One research postulated increasing sporulation by hypervirulent strain possibly has association with robust CDI spreading. Nevertheless, previous research demonstrated controverted results in regard to disease severity by hypervirulent strain. A retrospective study by Bauer et al.41 concluded hypervirulent strain BI/NAP1/027 as associated with declined odds of disease severity ratio (OR): 0.35, 95% confi dence interval (CI) 0.13 - 0.93) and did not increase mortality in hospitalized patients (OR: 1.02, 95% CI 0.53 - 1.96), or (OR: 1.16, 95% CI 0.36 - 3.77) of recurrence rate. Meanwhile, some other studies (cohort, case- control, and cross-sectional) did not demonstrate worse prognoses compared to other strains as shown in Table 1.41 IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA Share alike 4.0 33Ni Nyoman Sri Budayanti, et al.: Clostridium difficile Infection (CDI) by Hypervirulent Table 1. Virulence factors of hypervirulent BI/NAP1/027 C. diffi cile strain41 Virulence factors Mechanism TcdA or Enterotoxin A (Toxin A) Destruction of actin within target cells causes infi ltration of neutrophil, infl ammation, and epithelial cells necrosis. TcdB or Cytotoxin B (Toxin B) Destruction of epithelial cells tight junctions causes increasing permeability of vascular, and hemorrhage. CDTa toxin Modifi es the action with ADP-ribosylation leads to depolymerization of actin and damage of cytoskeleton assists bacteria adherence to epithelial cells of gut. CDTb toxin Facilitates CDTa toxin uptake into epithelial lining of gut. Hypersporulation Increases bacteria reproduction and spreading. Mutation of TcD gene Increases assembly of Toxin A and Toxin B by down-regulation of feedback inhibitor necessitate in diminishing toxin production. Based on Sirard et al. (2011), even though hypervirulent strain BI/NAP1/027 is able to assemble more toxins, they construct spores in fewer numbers and have not always been associated with severe condition of disease.42 In contrast, a cohort study by Rao et al.43 demonstrated association between hypervirulent strain ribotype 027 with severe CDI disease (OR: 1.73, 95% CI 1.03 - 2.89; p = 0.037) and higher mortality rate (OR: 2.02, 95% CI 1.19 - 3.43; p = 0.009) juxtaposed to other ribotypes.43 Study by See et al.44 demonstrated similar results by using NAP1 strain, where analysis of multivariate regression depicted increased severity of CDI (OR: 1.66, 95% CI: 1.90 - 2.54) and higher mortality (OR: 2.12, 95% CI: 1.22 - 3.68).44 Furthermore, a study in Quebec showed the hypervirulent strain ribotype 027 is associated with disease severity, twice more severe frequently in contrast to other strains. Nevertheless, basic reasons of these contradictory results were un-measured confounding factors, setting of study, detection methods of C. diffi cile, size of sample, population of study, and design of study. Therefore, the generalization of the study results has to be examined profoundly. Therefore, given these contrary fi ndings, healthcare workers or providers advised to center their attention on infection treatment based on clinical reasoning and infection marker related to severe infection, as well as episodes of diarrhea, dehydration signs, albumin level, creatinine level, white blood cell (WBC) count, underlying comorbidities, and immunocompromised condition.45,46 MECHANISM OF ENDEMIC STRAIN DISPLACEMENT WITH HYPERVIRULENT RIBOTYPE 027 C. diffi cile STRAIN Transmission of pathogen occur via fecal- oral route with new infections emerge by bacterial spore consumption. C. diffi cile spores are resistance to desiccation and able to persist for about 5 months on hard or solid surface. Merrigan et al.45 examined spore accumulation in regard to growth cycle of bacteria with results demonstrating that hypervirulent strains have the ability to sporulate faster and causing signifi cant more spore accumulation per total volume compared to non-hypervirulent strains.45 Increase sporulation rate could elucidate the uncommon soaring recurrence correlated to hypervirulent strains, 4-fold according to Marsh et al.20,46, as patients tend to transmit the contamination to local surroundings, then re-infect themselves subsequently. Subsequently after dormant bacterial consumption and ingestion, germination of C. diffi cile spore occurs as exposure to combination of bile salts and L-glycine. Vegetative phase of C. difficile happen as colonization of host’s gastrointestinal tract. Even tough colonization is required to cause the disease, most of infected people prevail asymptomatic. CDI manifestations are arbitrated by production of cytotoxic toxins to large intestine epithelial tissue lead the way of immense colon infl ammation and epithelial cell obstruction of the host.46 Study by Pepin et al.47 and Hubert et al.48 demonstrated doubling rate of severe disease as emergence of ribotype 027 in Canada. Hypervirulent strains associated IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA Share alike 4.0 34 Indonesian Journal of Tropical and Infectious Disease, Vol. 10 No. 1 January–April 2022: 27–41 with higher rates of symptomatic disease presumed to be result of increased production of toxin or due to intensifi ed variant clostridial toxins.48 There are three probable mechanisms postulated in accordance to transitions from endemic strains to hypervirulent strains: (1) the more infectious strains are hypervirulent; (2) symptomatic condition with higher rate is caused by hypervirulent strain; (3) outcompete in host’s gut can be done by hypervirulent strain.49 Throughout stochastic simulation, C. diffi cile hypervirulent strain invasion to human population cherished endemic strain was investigated. Reasoning of some previous models aims to establish infection control strategy in hospital and surroundings. Nevertheless, C. diffi cile has been recognized prominently as a global community pathogen, in preference to just segment of healthcare associated pathogen. In addition, present study has demonstrated major source of infection is community. Nonetheless, in some conditions if community not the primary source, infections suff ered by high-risk group in healthcare environment. Underlying cause of diff erence between endemic strains and hypervirulent strains prevail undetermined regardless of current atypical strains constitute predominant infections in community surroundings. Therefore, the consequence of three distinct pathways of intensifi ed were examined namely increasing pathogen infectiousness, increasing rate of colonization to symptomatic disease, and ability of endemic strains displacement by hypervirulent strains in colonized gut.49,52 Instinctively, parameters govern these distinct mechanisms have positive correlation to possibility of establishment invading strains in community. Nevertheless, comparison of these parameters’ infl uence on invasion rate and prevalence of resultant equipoise yielded different patterns of epidemiology. In accordance to classic epidemiological comprehension, the rate in which an establish pathogen spreading within susceptible individuals is strongly dependent on coeffi cient of transmission, as modelled by increasing the hypervirulent strain infectiousness. Simulation demonstrated increased infectious strains tend to establish further, spread robustly, and reach equilibrium to increased prevalence in community. Probability of successful established invasion and current steady circumstances of prevalence has been less influenced by rising colonized percentage on clinical disease experience. If individuals colonized by endemic strains were prone to hypervirulent strains, a weaker correlation was constructed with probability of establishment, and no comprehensible correlation was discerned with equilibrium prevalence outcome. Spreading of novel strain is substantially independent to endemicity of resident strain when gut is colonized by resident strain as uncolonized gut readily.49-53 Clinical reports over the past 15 years have demonstrated substantial increase of disease rate in accordance to prominent and robust switch in dominance of C. diffi cile strain. Isolates from PCR-ribotyping in Montreal hospital depicted NAP1/ribotype 027 were not found in 2000 and 2001. Nonetheless, NAP1/ribotype 027 constituted more than 75% isolates collected during the outbreak in 2003 until 2004. Increasing prevalence of CDI disease has corresponded to ribotype 027 dominance in many countries across the world, comprising England with its peak in 2007-2008, European countries, and North America.49,50 Tying to epidemiological model with present analysis results, apparently hypervirulent strains’ ability in displacing endemic strains from readily colonized host’s gut is the slightest mechanism facilitates ribotype 027 dominance, resulting in more severe diarrhea and longer recovery period. In spite of investigating a wide range of parameter values, from resistance of colonization to susceptibility counterpart in uncolonized individuals, novel strain is unsuccessful to reproduce heightened level of prevalence associated with emerging hypervirulent strains. It does not invalidate the probability of more competitive hypervirulent strains compared to typical strains within host. However, it still suggests this mechanism is not a pivotal role for successful invasion and hypervirulent strain of ribotype 027 clonal dominance. Importantly, the present study depicted strains’ competition inside host’s gut is not essential for abrupt prevailing strains switching; all surrogate mechanisms IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA Share alike 4.0 35Ni Nyoman Sri Budayanti, et al.: Clostridium difficile Infection (CDI) by Hypervirulent of hypervirulent distinctly illustrated previous dominant strains are not merely added on invasion of subsequent new strain, yet excluded throughout exploitative competition.53,54 A N T I B I O T I C R E S I S T A N C E O F HYPERVIRULENT BI/NAP1/027 C. diffi cile STRAIN Investigation of BI/NAP1/027 CDI cases in Panama showed high resistance to several antibiotics: rifampin, ciprofl oxacin, levofl oxacin, moxifloxacin, and clindamycin, yet remain susceptible to vancomycin and metronidazole. Study tested for several antibiotic susceptibility for ribotype 027 and non-027 ribotype in Canada with fi ndings 92.2% resistance of ribotype 027 to moxifl oxacin as opposed to 11.2% of other strains. Correspondingly, ribotype 027 strains (78.2%) showed resistance to ceftriaxone compared to other strains (15.7%). Ribotype 027 was greater than 4-fold higher of minimum inhibitory concentration (MIC) compared to metronidazole (4 μg/ml vs 1 μg/ml). In addition, ribotype 027 strain demonstrated 2-fold higher MIC of fidaxomicin (1 μg/ml vs 2 μg/ml). Nevertheless, resistance for vancomycin and clindamycin was akin both in group of BI/ NAP1/027 and other strains. Erythromycin resistance is associated with mutation of methylase genes in ribosome, meanwhile fl uoroquinolones resistance is caused by mutation of DNA gyrase. Resistance to fi daxomicin and rifamycin group is linked to methylation of ribonucleic acid (RNA) polymerase. In addition, resistance to linezolid is caused by genes of lincosamide and phenicol. Study in several hospitals in Mexico demonstrated numerous ribotype 027 isolates possesses decreased susceptibility to fi daxomicin even though this antibiotic is unavailable in Mexico and patients had been unexposed to it. Basis for BI/NAP1/027 strain treatment is antibiotics. Presently, specifi c guidelines of the Infectious Diseases Society of America (IDSA) remain unavailable to BI/NAP1/027 strain.55-58 Consequently, based on overall CDI treatment guidelines, infection by BI/NAP1/027 strain treatment has been proposed as in Table 2. Table 2. Suggestive antibiotic treatment for BI/NAP1/027 strain55 1st line treatment Alternative treatment Initial non-severe infection Vancomycin per oral (p.o.), 125 mg, 4 times daily, 10 days Fidaxomicin p.o., 200 mg, twice per day, 10 days. If unavailable, take metronidazole, 500 mg, three times per day, 10 days 1st non-severe recurrency Vancomycin p.o., 125 mg, 4 times per day, 10 days Oral fi daxomicin, 200 mg, twice per day, 10 days 2nd non-severe recurrency Vancomycin p.o. tapering off : 125 mg, 4 times, 7 until 10 days; 125 mg twice per day, 7 days; 125 mg once daily, 7 days; 125 mg per three days, 14 days Fidaxomicin p.o., 200 mg, twice per day, 10 days, or transplantation of fecal microbiota Later non-severe relapse Transplantation of fecal microbiota Vancomycin p.o. tapering off with probiotics, fi daxomicin, intravenous immune globulin (IVIG) Severe disease Vancomycin p.o. 125 mg, f4 times daily; rise to 500 mg, 4 times per day. This can be applied only if there is no improvement within 24 - 48 hours, or associated side eff ects, e.g., ileus, renal failure If patient cannot tolerate vancomycin p.o, fi daxomicin is antibiotic of choice Ileus Plus intravenous metronidazole 500 mg, every 8 hours to fi daxomicin or oral vancomycin, consultation to general surgery should be considered IVIG, intra colonic vancomycin IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA Share alike 4.0 36 Indonesian Journal of Tropical and Infectious Disease, Vol. 10 No. 1 January–April 2022: 27–41 HYPERVIRULENT BI/NAP1/027 C. diffi cile STRAIN PREVENTATIVE MEASURES AND CONTROL BI/NAP1//027 is well-known to cause outbreaks in hospital, and some reports have represented eff orts and measures in regard to outbreak control. Muto56 depicted combined measures to control outbreak in the University of Pittsburgh as a bundle of encompassed education; increment of early detection in regard to CDI requires nurses to make an order of toxin texting, and email notifi cation to alert physicians who treat high risk patients, and establish a management team to evaluate patients tested positive for all C. diffi cile toxins. Expansion of infection control action comprises environmental cleansing with bleach, replacing alcohol hand rubs with water and soap to improve hand hygiene for CDI patients, continuation of contact precautions following diarrhea resolution, audit of infection control, and restriction of targeted antimicrobials.56 Even though particular eff ect of each measures was diffi cult to ascertain, investigators delineated a 78% decrease of CDI incidence and severe CDI proportion. Furthermore, only 13% of C. diffi cile isolates were BI/NAP1/027 strain by 2005, compared to 51% among clinical isolates in 2001.57-60 In regard to numerous hospitals outbreak in Quebec, the Canadian government allocated $20 million to upgrade infection control measures in 12 hospitals; whereas in Pittsburgh, various approaches were implemented, comprising domestic measures intensifi cation with thorough environmental cleaning of aff ected areas and toilets by applying bleach, cleaning all rooms on a subdivision or section if number of nosocomial occurrence exceeded within three weeks; equipment dedication; applying hand washing rather than alcohol rubs; prompt finding of CDI case by daily enhancement of toxin testing frequency in clinical laboratory; prompt empirical treatment and contact precaution practice subsequent to second diarrhea stool; move patients from 4-bed ward if possible; and education to decrease administration of cephalosporin and fluoroquinolone. Consequently, incidence of CDI in these hospitals declined from 22.5 to 12.4 per 1000 admittance as a result of applying these preventative measures, but incidence rates did not reach pre-outbreak extent.61-65 One hospital in Quebec implemented antimicrobial stewardship when no effectivity was shown in decrease of CDI incidence after executing infection control measures. This unrestrained strategy leaned on education and commentary or assessment from pharmaceutical parties and hence attained administration reduction to 54% of total antibiotic and 23% of targeted antibiotic. Simultaneously, with diminishment in antibiotic consumption, CDI incidence has seen a 60% drop. Targeted antibiotics encompassed second and third generation of cephalosporin, macrolides, clindamycin, and ciprofl oxacin. Drop in ciprofl oxacin usage has been accompanied by increase. In other places, administration of moxifl oxacinwas used as an agent incriminated as high-risk antimicrobial agent.64,65 Some factors contribute a signifi cant role of therapy by fluoroquinolones in epidemics era, encompassing enhance resistance of BI/ NAP1/027 strains to group of fl uoroquinolones, juxtaposed to historical isolates not associated to epidemic isolates, expanded consumption of fl uoroquinolones, along with high ascribable risk in regard to fl uoroquinolones of this outbreak. However, considering assorted outcomes of certain fl uoroquinolones restriction, un-assessed hypothesis that could be a “class effect,” subsequently all fluoroquinolones restriction will be a specifi c potential control course of action in hospital with outbreak caused by BI/NAP1/027 strain. Various measures have been implemented in outbreak control of CDI, especially BI/NAP1/027 which poses a remarkable challenge.66-68 Coalescence of elderly patients, continual use of antibiotics, contamination of hospital environment with spores are all ideal circumstances of CDI outbreaks, high rate or number of morbidity and mortality. Even though infection control measures, such as environmental cleaning, isolation, and hand hygiene, will persist as keystones course of action to prevent C. diffi cile exposure in hospital, methods to reduce disease IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA Share alike 4.0 37Ni Nyoman Sri Budayanti, et al.: Clostridium difficile Infection (CDI) by Hypervirulent risk following C. diffi cile infection or ingestion have to be reckoned. Nevertheless, decreasing antibiotics use remains absolutely an important approach to reduce CDI risk. These measures have commenced in various hospitals, yet there is still considerable extent to improve antimicrobial stewardship. Methods to neutralize antibiotic disruption of microbiota colonization should be incorporated with biotherapeutic methods, e.g., nontoxigenic C. diffi cile strain administration which has demonstrated to be eff ective in hamster model. Focus measures also should emphasize on development of vaccine of C. difficile to boost immune state of elderly people. Passive immune methods such as monoclonal antibody to enhance immune response to toxin A and B seem to be eff ective in early stage of clinical trials. Nevertheless, even though current focus is on BI/NAP1/027 C. diffi cile strain, new upcoming epidemic strains are going to emerge in the foreseeable future.69-72 CONCLUSIONS Greater fatality and severity epidemics of CDI have been caused by highly virulent form of C. diffi cile of BI/NAP1/027 that spread widely and robustly over decades. Main risk factors are elderly age, hospitalization, and exposure to antibiotics, e.g., cephalosporins, fl uoroquinolones, clindamycin, and penicillin. Virulence factors are TcdA, TcdB, CDTa CDTb; hypervirulence is prone to hypersporulation and mutation of TcD gene leads to toxin hyperexpression. Preventative measures can be done by early cases detection, building a management team to evaluate patient positive with all C. diffi cile toxins, hand hygiene improvement, continuation of contact precautions after diarrhea resolution, audit of infection control, restriction of antimicrobials, and development of a vaccine of C. diffi cile. ACKNOWLEDGEMENT We would like to acknowledge Clinical Microbiology Department and Geriatric Division, Internal Medicine Department, Faculty of Medicine, Universitas Udayana to support this literature review. CONFLICT OF INTEREST No competing interests have been associated from construction process until publication of the manuscript. No fi nancial support from any parties or organization was obtained regarding the manuscript submitted. REFERENCES 1. Kotila SM, Mentula S, Ollgren J, Virolainen- Julkunen A, Lyytikäinen O. Community- and Healthcare-Associated Clostridium diffi cile Infections, Finland, 2008−2013. Emerging Infectious Disease journal. [Online] 2016;22(10): 1747. Available from: doi:10.3201/eid2210.151492 2. Banaei N, Anikst V, Schroeder LF. Burden of Clostridium diffi cile infection in the United States. [Online] The New England Journal of Medicine. United States; 2015. p. 2368–2369. Available from: doi:10.1056/NEJMc1505190#SA2 3. Lucado J, Gould C, Elixhauser A. Clostridium Diffi cile Infections (CDI) in Hospital Stays, 2009: Statistical Brief #124. Rockville (MD); 2006. 4. Collins DA, Hawkey PM, Riley T V. Epidemiology of Clostridium diffi cile infection in Asia. Antimicrobial resistance and infection control. [Online] England; 2013;2(1): 21. Available from: doi:10.1186/2047- 2994-2-21 5. Putsathit P, Maneerattanaporn M, Piewngam P, Kiratisin P, Riley T V. Prevalence and molecular epidemiology of Clostridium diffi cile infection in Thailand. New Microbes and New Infections. [Online] Elsevier Ltd; 2017;15: 27–32. Available from: doi:10.1016/j.nmni.2016.10.004 6. Rupnik M, Grabnar M, Geric B. Binary toxin producing Clostridium diffi cile strains. Anaerobe. [Online] England; 2003;9(6): 289–294. Available from: doi:10.1016/j.anaerobe.2003.09.002 7. Oyofo BA, Subekti D, Tjaniadi P, Machpud N, Komalarini S, Setiawan B, et al. Enteropathogens associated with acute diarrhea in community and hospital patients in Jakarta, Indonesia. FEMS immunology and medical microbiology. England; 2002;34(2): 139–146. 8. Collins DA, Gasem MH, Habibie TH, Arinton IG, Hendriyanto P, Hartana AP, et al. Prevalence and molecular epidemiology of Clostridium difficile infection in Indonesia. New Microbes and New Infections. [Online] Elsevier Ltd; 2017;18: 34–37. Available from: doi:10.1016/j.nmni.2017.04.006 IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA Share alike 4.0 38 Indonesian Journal of Tropical and Infectious Disease, Vol. 10 No. 1 January–April 2022: 27–41 9. Rupnik M. Clostridium difficile : (Re)emergence of Zoonotic Potential. Clinical Infectious Diseases. [Online] 2010;51(5): 583–584. Available from: doi:10.1086/655693 10. Kyne L, Warny M, Qamar A, Kelly CP. Asymptomatic carriage of Clostridium diffi cile and serum levels of IgG antibody against toxin A. The New England journal of medicine. [Online] United States; 2000;342(6): 390–397. Available from: doi:10.1056/ NEJM200002103420604 11. Kyne L, Warny M, Qamar A, Kelly CP. Association between antibody response to toxin A and protection against recurrent Clostridium difficile diarrhoea. Lancet (London, England). [Online] England; 2 0 0 1 ; 3 5 7 ( 9 2 5 1 ) : 1 8 9 – 1 9 3 . Av a i l a b l e f r o m : doi:10.1016/S0140-6736(00)03592-3 12. Pepin J, Saheb N, Coulombe M-A, Alary M-E, Corriveau M-P, Authier S, et al. Emergence of fl uoroquinolones as the predominant risk factor for Clostridium diffi cile-associated diarrhea: a cohort study during an epidemic in Quebec. Clinical infectious diseases : an offi cial publication of the Infectious Diseases Society of America. [Online] United States; 2005;41(9): 1254–1260. Available from: doi:10.1086/496986 13. Johnson S. Recurrent Clostridium diffi cile infection: a review of risk factors, treatments, and outcomes. The Journal of infection. [Online] England; 2009;58(6): 403–410. Available from: doi:10.1016/j. jinf.2009.03.010 14. Hoyert DL, Xu J. Deaths: preliminary data for 2011. National vital statistics reports : from the Centers for Disease Control and Prevention, National Center for Health Statistics, National Vital Statistics System. [Online] 2012;61(6): 1–51. Available from: http:// www.ncbi.nlm.nih.gov/pubmed/24984457 15. Khanna S, Pardi DS, Aronson SL, Kammer PP, Orenstein R, St Sauver JL, et al. The epidemiology of community-acquired Clostridium diffi cile infection: a population-based study. The American journal of gastroenterology. [Online] United States; 2012;107(1): 89–95. Available from: doi:10.1038/ajg.2011.398 16. Dumyati G, Stevens V, Hannett GE, Thompson AD, Long C, MacCannell D, et al. Community-associated Clostridium difficile Infections, Monroe County, New York, USA. Emerging Infectious Diseases. [Online] Centers for Disease Control and Prevention; 2012;18(3): 392–400. Available from: doi:10.3201/ eid1803.102023 17. Khurana A, Vinayek N, Recco RA, Go ES, Zaman MM. The incidence of Clostridium diffi cile-associated and non-C. diffi cile-associated diarrhea after use of gatifl oxacin and levofl oxacin in an acute-care facility. [Online] Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. United States; 2004. p. 602–603. Available from: doi:10.1086/422525 18. Ho J, Dai RZW, Kwong TNY, Wang X, Zhang L, Ip M, et al. Disease Burden of Clostridium diffi cile Infections in Adults, Hong Kong, China, 2006-2014. Emerging infectious diseases. [Online] 2017;23(10): 1671–1679. Available from: doi:10.3201/eid2310.170797 19. Kim YS, Han DS, Kim YH, Kim WH, Kim JS, Kim HS, et al. Incidence and clinical features of Clostridium diffi cile infection in Korea: a nationwide study. Epidemiology and infection. [Online] England; 2013;141(1): 189–194. Available from: doi:10.1017/ S0950268812000581 20. Marsh JW, Arora R, Schlackman JL, Shutt KA, Curry SR, Harrison LH. Association of Relapse of Clostridium difficile Disease with BI/NAP1/027. Journal of Clinical Microbiology. [Online] 1752 N St., N.W., Washington, DC: American Society for Microbiology; 2012;50(12): 4078–4082. Available from: doi:10.1128/JCM.02291-12 21. Kyne L, Hamel MB, Polavaram R, Kelly CP. Health care costs and mortality associated with nosocomial diarrhea due to Clostridium difficile. Clinical infectious diseases. [Online] 2002;34(3): 346–353. Available from: doi:10.1086/338260 22. Jones AM, Kuijper EJ, Wilcox MH. Clostridium difficile: a European perspective. The Journal of infection. [Online] England; 2013;66(2): 115–128. Available from: doi:10.1016/j.jinf.2012.10.019 23. Sitzlar B, Vajravelu RK, Jury L, Donskey CJ, Jump RLP. Environmental decontamination with ultraviolet radiation to prevent recurrent Clostridium diffi cile infection in 2 roommates in a long-term care Facility. [Online] Infection control and hospital epidemiology. United States; 2012. p. 534–536. Available from: doi:10.1086/665310 24. Guerrero DM, Nerandzic MM, Jury LA, Jinno S, Chang S, Donskey CJ. Acquisition of spores on gloved hands after contact with the skin of patients with Clostridium diffi cile infection and with environmental surfaces in their rooms. American journal of infection control. [Online] United States; 2012;40(6): 556–558. Available from: doi:10.1016/j.ajic.2011.08.002 25. Asempa TE, Nicolau DP. Clostridium diffi cile infection in the elderly: an update on management. Clinical Interventions in Aging. [Online] Dove Medical Press; 2017;12: 1799–1809. Available from: doi:10.2147/ CIA.S149089 26. Cohen SH, Gerding DN, Johnson S, Kelly CP, Loo VG, McDonald LC, et al. Clinical practice guidelines for Clostridium diffi cile infection in adults: 2010 update by the society for healthcare epidemiology of America (SHEA) and the infectious diseases society of America (IDSA). Infection control and hospital IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA Share alike 4.0 39Ni Nyoman Sri Budayanti, et al.: Clostridium difficile Infection (CDI) by Hypervirulent epidemiology. [Online] United States; 2010;31(5): 431–455. Available from: doi:10.1086/651706 27. Leffler DA, Lamont JT. Clostridium difficile Infection. New England Journal of Medicine. [Online] Massachusetts Medical Society; 2015;372(16): 1539– 1548. Available from: doi:10.1056/NEJMra1403772 28. Procop G, Church D, Hall G, Janda W, Koneman E, Schreckenberger P, et al. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology.. Seventh Ed. Joyce J (ed.) Philadelphia: Wolters Kluwer; 2017. 29. Mulvey MR, Boyd DA, Gravel D, Hutchinson J, Kelly S, McGeer A, et al. Hypervirulent Clostridium diffi cile strains in hospitalized patients, Canada. Emerging Infectious Diseases. [Online] 2010;16(4): 678–681. Available from: doi:10.3201/eid1604.091152 31. Sachsenheimer FE, Yang I, Zimmermann O. Genomic and phenotypic diversity of Clostridium diff cile during long-term sequential recurrence of infection. Int J Med Microbiol. 2018;308: 364-77. Available from: doi:10.1016/j.ijmm.2018.02.002 32. Luciano JA, Zuckerbraun BS. Clostridium diffi cile infection: prevention, treatment, and surgical management. Surg Clin North Am. 2014;94: 1355-49. Available from: doi:10.1016/j.suc.2014.08.006 33. Clabots CR, Johnson S, Olson MM, Peterson LR, Gerding DN. Acquisition of Clostridium diffi cile by hospitalized patients: evidence for colonized new admissions as a source of infection. J Infect Dis. 1992;166: 561-67. Available from: https://www.jstor. org/stable/30113322 34. Howell M, Novack V, Grgurich P, Soulliard D, Novack L, Pendina M, Talmor D. Iatrogenic gastric acid suppression and the risk of nosocomial Clostridium diffi cile infection. Arch Intern Med. 2010;170: 784-90. Available from: doi:10.1001/archinternmed.2010.89 35. O’Keefe S. Tube feeding, the microbiota, and Clostridium diffi cile infection. World J Gastroenterol. 2010;16: 139-42. Available from: doi:10.3748/wjg. v16.i2.139 36. Hampton T. Report reveals scope of US antibiotic resistance threat. JAMA. 2013;310: 1661-63. Available from: doi: 10.1001/jama.2013.280695 37. Popoff MR, Rubin EJ, Gill DM. Actin-specifi c ADP- ribosyltransferase produced by a Clostridium diffi cile strain. Infect Immun. 1998;56: 2299-2306. Available from: doi: 10.1128/iai.56.9.2299-2306.1988 38. Jamal W, Rotimi VO, Brazie J, Duerden BI. Analysis of prevalence, risk factors and molecular epidemiology of Clostridium diffi cile infection in Kuwait over a 3-year period. Anaerobe. 2010;16: 560-65. Available from: doi:10.1016/j.anaerobe.2010.09.003 39. Jalali M, Khorvash F, Warriner K, Weese J. Clostridium diffi cile infection in an Iranian hospital. BMC Res Notes. 2012;5: 159. Available from: 10.1186/1756- 0500-5-159 40. Al-Thani AA, Hamdi WS, Al-Ansari NA, Doiphode SH, Wilson GJ. Polymerase chain reaction ribotyping of Clostridium diffi cile isolates in Qatar: a hospital- based study. BMC Infect Dis. 2014;14: 502. Available from: doi:10.1186/1471-2334-14-502 41. Bauer KA, Johnston JEW, Wenzler E. Impact of the NAP-1 strain on disease severity, mortality, recurrence of healthcare-associated Clostridium diffi cile infection. Anaerobe. 2017;48: 1-6. Available from: doi:10.1016/j.anaerobe.2017.06.009 42. Sirard S, Valiuette L, Fortier LC. Lack of asscociation between clinical outcome of Clostridium diffi cile infetions, strain type, and virulence-associated phenotypes. J Clin Microbiol. 2011;49: 4040-46. Available from: doi:10.1128/jcm/05053-11 43. Rao K, Micic D, Natarajan M. Clostridium diffi cile ribotype 027: relationship to age, detectability of toxins A or B in stool with rapid testing, severe infection, and mortality. Clin Infect Dis. 2015;61: 233-41. Available from: doi:10.1093/cid/civ254 44. See I, Mu Y, Cohen J. NAP1 strain type predicts outcomes from Clostridium diffi cile infection. Clin Infect Dis. 2014;58: 1394-400. Available from: doi:10.1093/cid/ciu125 45. Merrigan M, Sambol S. Johnson. Susceptibility to hamsters to human pathogenic Clostridium diffi cile strain B1 following clindamycin, ampicillin or ceftriaxone administration. Anaerobe. 2003;9: 9 1 - 9 5 . Av a i l a b l e f r o m : d o i : 1 0 . 1 0 1 6 / s 1 0 7 5 - 9964(03)00063-5 46. Marsh JW. Association of relapse of Clostridium diffi cile disease with BI/NAP1/027. J Clin Microbiol. 2012;50: 4078-4082. Available from: doi:10.1128/ jcm.02291-12 47. Pepin J, Valiquette L, Alary ME. Clostridium diffi cile- associated diarrhea in a region of Quebec from 1991 to 2003: a changing pattern of disease severity. CMAJ. 2004;171: 466-472. Avalaible from: doi:10.1503/ cmaj.1041104 48. Hubert B, Loo VG, Bourgault AM. A portrait of the geographic dissemination of the Clostridium diffi cile North American pulsed-fi eld type 1 strain and the epidemiology of C. difficile-assocated disease in Quebec. Clin Infect Dis. 2007;44: 238-44. Available from: doi:10.1086/510391 49. Yakob L, Riley TV, Paterson DL, Marquess J, Magalhaes RJS, Kanamori LF, Clements ACA. Mechanisms of hypervirulent Clostridium difficile ribotype 027 displacement of endemic: an epidemiological model. Nature Scientifi c Report. 2019;5: 12666. Available from: doi: 10.1038/srep12666. 50. Cheng V, Yam W, Lam O. Clostridium diffi cile isolates with increased sporulation: emergence of PCR ribotype 002 in Hong Kong. Eur J Clin Microbiol Infect Dis. infection in the community: a zoonotic disease? Clin Microbiol Infect. 2012;18: 635-45. Available from: doi: 10.1111/j.1469-0691.2012.03853.x 30. Hensgens MP, Keessen EC, Squire MM. Clostridium difficile IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA Share alike 4.0 40 Indonesian Journal of Tropical and Infectious Disease, Vol. 10 No. 1 January–April 2022: 27–41 2011;30: 1371-81. Available from: doi:10.1007/ s10096-011-1231-0 51. Kim H, Lee Y, Moon H, Lim C, Lee K, Chong Y. Emergence of Clostridium diffi cile ribotype 027 in Korea. Korean J Lam Med. 2011;31: 191-96. Available from:doi: https://dx.doi.org/10.3343%2Fkjlm.2011.3 1.3.191 52. Krutova M, Nyc O, Matejkova J, Kuijper E, Jalava I, Mentula S. The recognition and characterisation of Finnish Clostridium diff cile isolates resembling PCR-ribotype 027. J Microbiol Immunol Infect. 2018;51: 344-51. Available from: doi:10.1016/j. jmii.2017.02.002 53. Freeman J, Vernon J, Pilling S. The CloseER study: results from a three-year pan-European longitudinal surveillance of antibiotic resistance among prevalent Clostridium difficile ribotype, 2011-2014. Clin Microbiol Infect. 2018;24: 724-31. Available from: doi:10.1016/j.cmi.2017.10.008 54. Giancola S, Williams R, Gentry C. Prevalence of Clostridium diffi cile BI/NAP1/027 strain across the United States Veterans Health Administration. Clin Microbiol Infect. 2018;24: 877-81. Available from: 10.1016/j.cmi.2017.11.011 55. Pituch H, Obuch-Woszczatynski P, Lachowiz D. Prevalence of Clostridium diffcile infection in hospitalized patients with diarhrhoea: results of a Polish multicenter, prospective, biannual point- prevalence study. Adv Med Sci. 2018;63: 290-95. Available from: doi:10.1016/j.advms.2018.03.003 56. Muto CA, Blank MK, Marsh JW. Control of an outbreak of infection with hypervirulent Clostridium diffi cile BI strain in a university using comprehensive “bundle” approach. Clin Infect Dis. 2007;45: 1266- 1273. Available from: doi:10.1086/522654 56. DePestel DD, Aronoff DM. Epidemiology of Clostridium diffi cile infection. J Pharm Pract. 2013;26: 464-75. Available from: doi: 10.1177/0897190013499521 57. Gerding DN, Johnson S, Rupnik M, Aktories K. Clostridium diffi cile binary toxin CDT: mechanism, epidemiology, and potential clinical importance. Gut Microbes. 2014;5: 15-27. Available from: doi: 10.4161/gmic.26854 58. Warny M, Pepin, Fang A. Toxin production by an emerging strain of Clostridium diff cile associated with outbreaks of severe disease in North America and Europe. Lancet. 2005;366: 1079-84. Available from: doi: 10.1016/S0140-6736(05)67420-X 59. Akerlund T, Persson I, Unemo M, Noren T, Svenungsson B, Wult M, Burman LG. Increased sporulation rate of epidemic Clostridium diffi cile type 027/NAP1. J Clin Microbiol. 2008;46: 1530-33. Availabe from: doi: 10.1128/JCM.01964-07 60. Cloud J, Noddin L, Pressman A, Hu M, Kelly C. Clostridium diff cile strain NAP-1 is not associated with severe disease in a nonepidemic setting. Clin Gastroeneterol Hepatol. 2009;7: 868-873.e2. Available from: doi: 10.1016/j.cgh.2009.05.018 61. Morgan OW, Rodrigues B, Elston T, Verlander NQ, Brown DF, Brazier J, Reacher M. Clinical severity of Clostridium diffi cile PCR ribotype 027: a case- study. PLoS One. 2008;3: e1812-10. Availabe from: doi:10.1371/journal.pone.0001812 63. Walk ST, Mimic D, Jain R. Clostridium difficile ribotype does not predict severe infection. Clin Infect Dis. 2012; 55: 1661-68. Available from: doi:10.1093/ cid/cis786 64. Sirard S, Valiquette L, Forties LC. Lack of association bertween clinical outcome of Clostridium diffi cile infections, strains type, and virulence-associated phenotypes. J Clin Microbiol. 2011; 49:4040-46. Availabe from: doi:10.1128/jcm.05053-11 65. Hsu J, Abad C, Dinh M, Safdar N. Prevention of endemic healthcare-associated Clostridium diffi cile infection: reviewing the evidence. Am J Gastroenterol. 2010;105: 2327-39. Available from: doi:10.1038/ ajg.2010.254 66. Secore S, Wang S, Doughtry J. Development of a novel vaccine containing binary toxin for prevention of Clostridium diff cile disease with enhanced effi cacy against NAP1 strains. PLoS One. 2017;12: e0170640. Available from: doi:10.1371/journal.pone.0170640 67. Kokai-Kun JF, Roberts T, Coughlin O. The oral β-lactamase SYN-004 (ribaxamase) degrades ceftriaxone excreted into the intestine in phase 2a clinical studies. Antimicrob Agents Chemother. 2017;61: e02197-16. Available from: doi:1128/ aac.02197-16 68. Karlowsky JA, Adam HJ, Kosowan T. PCR ribotyping and antimicrobial susceptibility testing of isolates of Clostridium diffi cile cultured from toxin-positive diarrheal stools of patients receiving medical care in Canadian hospitals: the Canadian Clostridium diffi cile Surveillance Study (CAN-DIFF) 2013-2015. Diagn Microbiol Infect Dis. 2018;91: 105-11. Available from: doi:10.1016/j.diagmicrobio.2018.01.017 69. Vickers RJ, Tillotson GS, Nathan R. Effi cacy and safety and ridinilazole compared with vancomycin for the treatment of Clostridium diffi cile infection: a phase 2, randomised, double-blind, active- controlled, and non-inferiority study. Lancet Infect Dis. 2017;17: 735-44. Available from: doi:10.1016/ s1473-3099(17)30235-9 70. Coff man K, Chen XJC, Okamura C, Louie E. IVIG - A cure to severe refractory NAP-1 Clostridium diffi cile colitis? A case of successful treatment of severe infection, which failed standard therapy including fecal microbiota transplants and fi daxomicin. IDCases. 2017;8: 27-28. Available from: doi:10.1016/j. idcr.2017.03.002 IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA Share alike 4.0 41Ni Nyoman Sri Budayanti, et al.: Clostridium difficile Infection (CDI) by Hypervirulent 71. McDonald LC, Gerding DN, Johnson S. Clinical practice guidelines for Clostridium diffi cile infection in adults and children: 2017 update by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA). Clin Infect Dis. 2018;66: e1-e48. Available from: doi:10.1093/cid/cix1085 72. Sawabe E, Kato H, Osawa K, Chida T, Tojo N, Arakawa Y, Okamura N. Molecular analysis of Clostridium diffi cile at a university teaching hospital in Japan: a shift in the predominance type over a fi ve-year period. Eur J Clin Microbiol Infect Dis. 2007;26: 695-703. Available from: doi: 10.1007/s10096-007-0355-8 IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA Share alike 4.0