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Clostridium difficile infection (CDI) in children due to hypervirulent strains PCR-ribotype 027: An emblematic report of two cases
Anaerobe xxx (2015) 1e3
Contents lists available at ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Clostridium difficile infection (CDI) in children due to hypervirulent strains PCR-ribotype 027: An emblematic report of two cases Patrizia Spigaglia a, *, Fabrizio Barbanti a, Elio Castagnola b, Roberto Bandettini c , Rome, Italy Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanita Infectious Diseases Unit, Istituto Giannina Gaslini, Genoa, Italy c Clinical Pathology Laboratory Unit, Istituto Giannina Gaslini, Genoa, Italy a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 July 2015 Received in revised form 10 September 2015 Accepted 15 September 2015 Available online xxx
In this report, the first two cases of pediatric Clostridium difficile infection (CDI) due to the hypervirulent PCR-ribotype 027 in Italy are described as emblematic of the role of both the infecting C. difficile strain and patient status in the occurrence and clinical manifestation of CDI in children. © 2015 Elsevier Ltd. All rights reserved.
Keywords: C. difficile Children PCR-ribotype 027 HSCT GVHD Antibiotics
1. Introduction An increased incidence of Clostridium difficile infection (CDI) has been recently observed in pediatric patients, particularly in North America where PCR-ribotype 027 (North American pulsed-field type 1, NAP1) has been recognized as cause of nosocomial diarrhea in 10e24% of symptomatic children [1,2]. Hypervirulent PCRribotype 027 has emerged in the last decade and it is now endemic throughout the U.S., Canada, and Europe [3]. C. difficile strains 027 show resistance to fluoroquinolones, production of binary toxin (CDT) and alterations of the tcdC gene, coding for the negative toxins regulator. This PCR-ribotype, associated with increased morbidity and mortality of infection in adults may be contributing to increasing pediatric CDI rates as well. In Italy, PCR-ribotype 027 is emerging and it actually represents the 9% of the toxigenic strains isolated in hospitals [4]. In this report, we described the first two cases of CDI by PCR-ribotype 027
* Corresponding author. Department of Infectious, Parasitic and ImmuneMediated Diseases, Istituto Superiore di Sanit a, Viale Regina Elena 299, 00161 Rome, Italy. E-mail address: [email protected] (P. Spigaglia).
in Italy as emblematic of the role of both the infecting C. difficile strain and patient status in the occurrence and clinical manifestation of infection.
2. Case one A 17-year-old female was hospitalized in October 2014. The patient was in immunosuppressive therapy for chronic graftversus-host disease (GVHD), following allogeneic hemopoietic stem cell transplant (allo-HSCT) for acute leukemia. The patient, with chronic lung disease due to GVHD and a Pseudomonas aeruginosa chronic infection, was undergoing to antibiotic treatment with piperacillin-tazobactam and tobramycin. She was treated empirically with ciprofloxacin because of a worsening of respiratory disease in another structure, located in a different Italian region, and then transferred in the Gaslini hospital. At time of admission, the patient presented abdominal pain and distension, diarrhea (5 lousy stools/day), temperature of 36 C, pulse rate of 68 beats/min, respiratory rate of 24/min, blood pressure of 125/83 mm Hg and oxygen saturation of 98%. Patient's clinical history reported an episode of C. difficile intestinal disease by a toxigenic strain in December 2012. In three following hospitalizations in 2013, the
http://dx.doi.org/10.1016/j.anaerobe.2015.09.004 1075-9964/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: P. Spigaglia, et al., Clostridium difficile infection (CDI) in children due to hypervirulent strains PCR-ribotype 027: An emblematic report of two cases, Anaerobe (2015), http://dx.doi.org/10.1016/j.anaerobe.2015.09.004
2
P. Spigaglia et al. / Anaerobe xxx (2015) 1e3
screening for C. difficile was always negative. After admission in our hospital, laboratory analysis revealed a white blood cell count (WBC) of 6.9 103/ml with 92.8% of neutrophils, fecal samples were negative for Adenovirus, Rotavirus, Cryptosporidium and Helicobacter pylori tests but positive for presumptive C. difficile PCRribotype 027 using the Xpert® C. difficile (Cepheid), according to manufacturer guidelines. The patient was treated for CDI with vancomycin (125 mg/6 h) for ten days. The clinical picture was further complicated for the development of a gastrointestinal tract graft-versus-host disease (GI GVDH) with presence of diarrhea (up to 7 lousy stools/day) and absence of fever. The patient was discharged at the 13th day of hospitalization. 3. Case two A 7-year-old male patient was hospitalized in October 2014. The patient was previously admitted in another hospital of the same Italian region and, after two days, he was transferred in the Gaslini hospital for abdominal pain. No clinical history of previous underlying disease. At time of admission, the patient presented a clinical picture of acute abdomen, temperature of 36 C, pulse rate of 102 beats/min, respiratory rate of 22/min and blood pressure of 121/ 73 mm Hg. An initial diagnosis of acute appendicitis was made and ampicillin-sulbactam and metronidazole were administered as therapy. The day after admission, the patient underwent surgery for acute appendicitis. The day after surgery, the patient presented a mild diarrhea (2 lousy stools in 12 h). Laboratory analysis revealed a WBC of 10.7 103/ml with a differential of 77.8% of neutrophils and C-RP of 9.18 mg/dl. Fecal cultures were negative for Salmonella spp, Shigella spp and Campylobacter spp. but positive for presumptive C. difficile PCR-ribotype 027 using the Xpert® C. difficile (Cepheid), according to manufacturer guidelines. Fecal sample was negative to Rotavirus, Adenovirus and Norovirus tests. The patient was treated with metronidazole (30e40 mg/kg/die) and vancomycin (40 mg/ kg/die). The body temperature was always around 36 C, except on the third day after surgery (37.9 C). Three days after the beginning of treatment the clinical picture improved (WBC of 10 103/ml, 70.5% of neutrophils and C-RP of 1.47 mg/dl) and the patient had formed stool on the fourth day. The patient was discharged from the hospital 11 days after the admission, in good clinical conditions (temperature of 36.4 C, WBC of 10.3 103/ml, 64.2% of neutrophils and C-RP < 0.46 mg/dl). 4. Detection, typing and characterization of strains In both cases, fecal samples were inoculated on Brucella Blood Agar and plates were incubated for 48 h at 37 C in Bio-Bag™ Environmental Chambers (Becton Dickinson) for anaerobic bacteria culture. Culture plates were sent to the Istituto Superiore di Sanita for C. difficile typing and analysis of antibiotic susceptibility. Briefly, C. difficile strains were isolated on the selective plates chromID™ rieux) after 48 h of incubation at 35 C under C. difficile (bioMe anaerobic conditions (85% N2, 10% H2 and 5% CO2). Results obtained by Xpert® C. difficile (Cepheid) were confirmed by C. difficile typing using the capillary gel electrophoresis PCR-ribotyping method, as described by Indra et al. [5]. PCR-ribotypes were determined by submitting data to the free web-database (http://webribo.ages.at). The minimum inhibitory concentrations (MICs) for moxifloxacin (MXF), erythromycin (ERY), clindamycin (CLI), rifampicin (RIF), metronidazole (MTZ) and vancomycin (VAN) were determined by Agar Dilution method on Brucella agar plates containing 1 mg/L vitamin K, 5 mg/L haemin and 5% defibrinated sheep red blood cells. In accordance with the Clinical and Laboratory Standards Institute (CLSI) interpretative categories approved for anaerobic bacteria the breakpoints for MXF and CLI was 8 mg/L, whereas the
breakpoint for MTZ was 32 mg/L [6]. The breakpoints for RIF, ERY and VAN were 4, 8 and 16 mg/L, respectively, in accordance with the CLSI interpretive categories approved for Staphylococcus aureus [6], since no values were provided for anaerobes. 5. Discussion The first two pediatric cases of CDI due to PCR-ribotype 027 in Italy occurred in a pediatric hospital located in the north of the Country. Both patients were transferred in the hospital from other structures. No epidemiologic evidence of patient-to-patient transmission of the same strain was found, and no outbreaks or cases of diarrhea occurred during the period in which the two patients were recovered. Unfortunately, no C. difficile epidemiological data are available about the hospitals in which the patients were firstly admitted. In this study, both patients did not show clinically signs and symptoms compatible with a severe CDI, as defined by the European Society of Clinical Microbiology and Infectious Diseases [7], even if infected by a hypervirulent strain. It has been observed that C. difficile PCR-ribotype does not always can be considered predictive of severe disease [8]. Furthermore, a recent study performed in U.S.A. indicate few markers of severe disease of CDI in HSCT pediatric patients, also if infected by strains 027 [9], probably because immunosuppression may attenuate the disease. Several risk factors for the development of CDI are known, first of all medication administration (e.g., antibiotics, acid suppression medication, and chemotherapy) [10,11]. In adults, inflammatory bowel disease (IBD) and human stem cell transplantation have been associated with CDI [12,13], whereas in children solid tumors, organ transplantation, gastrostomy, or jejunostomy represent a further risk for this infection [14,15]. In the cases here described, both patients were subject to antibiotics therapy before developing CDI: patient of case one was treated with ciprofloxacin due to a chronic lung GVHD and a P. aeruginosa infection, while patient of case two has a perioperative treatment with ampicillin-sulbactam plus metronidazole. As known, fluoroquinolones have been significantly associated to CDI due to strains 027 [16]. Both strains analyzed were multi-drug resistant (MDR) with high MIC to MXF (32 mg/L), ERY (MIC 256 mg/L), RIF (MIC 32 mg/L) and MIC ¼ 8 mg/L to CLI. Both strains were susceptible to MTZ and VAN, with MIC ¼ 0.25 mg/ L for both antibiotics. In recent papers, high geometric mean of MICs to MTZ of strains 027 [17] and cases of treatment failures of CDI cause by PCR-ribotype 027 after therapy with MTZ have been described [18]. In this study, analysis of antibiotic susceptibility was performed after freezing and several passages on culture plates that can cause MIC values reduction to MTZ, as previously observed [19]. Interestingly, when strains were subjected to sub-concentrations of MTZ, some colonies reached a MIC ¼ 8 mg/L (data not shown). Therefore, the capability to survive in vivo in presence of multiple antibiotics, including MTZ, play a key role in the occurrence of CDI independently from the status of the patient. This report describe two emblematic cases of CDI due to PCRribotype 027 in children. Clinical manifestations of CDI in these patients was not severe, despite the characteristics of hypervirulence of the infecting C. difficile strains. Anyway, strain characteristics have probably a crucial role in survival of the pathogen and occurrence of infection. Pediatric population subgroups, including HSCT patients and those who received multiple classes of antibiotics, are more susceptible to CDI. HSCT children seem to be particularly prone to CDI in the first year after transplantation, as observed in adults [20]. These patients can undergo to CDI recurrences and an association between CDI and GI GVDH seems to be possible. All these features should be considered with particular
Please cite this article in press as: P. Spigaglia, et al., Clostridium difficile infection (CDI) in children due to hypervirulent strains PCR-ribotype 027: An emblematic report of two cases, Anaerobe (2015), http://dx.doi.org/10.1016/j.anaerobe.2015.09.004
P. Spigaglia et al. / Anaerobe xxx (2015) 1e3
attention to promptly identify and control infections due to 027 in children, especially in countries like Italy where this PCR-ribotype is currently expanding. References [1] A. Deshpande, C. Pant, M.P. Anderson, C.J. Donskey, T.J. Sferra, Clostridium difficile infection in the hospitalized pediatric population: increasing trend in disease incidence, Pediatr. Infect. Dis. J. 32 (2013) 1138e1140. [2] P. Toltzis, J. Kim, M. Dul, J. Zoltanski, S. Smathers, T. Zaoutis, Presence of the epidemic North American pulsed field type 1 Clostridium difficile strain in hospitalized children, J. Pediatr. 154 (2009) 607e608. [3] M. Warny, J. Pepin, A. Fang, G. Killgore, A. Thompson, J. Brazier, E. Frost, L.C. McDonald, Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe, Lancet 366 (2005) 1079e1084. [4] P. Spigaglia, F. Barbanti, M. Morandi, M.L. Moro, P. Mastrantonio, Aspetti microbiologici delle infezioni da Clostridium difficile in Italia: risultati di uno studio condotto negli anni 2012e2013, Microbiol. Medica 29 (2014) 4722. [5] A. Indra, S. Huhulescu, M. Schneeweis, P. Hasenberger, S. Kernbichler, A. Fiedler, G. Wewalka, F. Allerberger, E.J. Kuijper, Characterization of Clostridium difficile isolates using capillary gel electrophoresis-based PCR ribotyping, J. Med. Microbiol. 57 (2008) 1377e1382. [6] Clinical and Laboratory Standards Institute, Performance Standards for Antimicrobial Susceptibility Testing, 24th Informational Supplement M100-S24, CLSI, Wayne, PA, USA, 2014. [7] S.B. Debast, M.P. Bauer, E.J. Kuijper, European Society of Clinical Microbiology and Infectious Diseases, European Society of Clinical Microbiology and Infectious Diseases: update of the treatment guidance document for Clostridium difficile infection, Clin. Microbiol. Infect. 20 (2014) 1e26. [8] S.T. Walk, D. Micic, R. Jain, E.S. Lo, I. Trivedi, E.W. Liu, L.M. Almassalha, S.A. Ewing, C. Ring, A.T. Galecki, M.A. Rogers, L. Washer, D.W. Newton, P.N. Malani, V.B. Young, D.M. Aronoff, Clostridium difficile ribotype does not predict severe infection, Clin. Infect. Dis. 55 (2012) 1661e1668. [9] M. Kamboj, K. Xiao, A. Kaltsas, Y.T. Huang, J. Sun, D. Chung, S. Wu, A. Sheahan, K. Sepkowitz, A.A. Jakubowski, G. Papanicolaou, Clostridium difficile infection after allogeneic hematopoietic stem cell transplant: strain diversity and outcomes associated with NAP1/027, Biol. Blood Marrow Transpl. 20 (2014) 1626e1633.
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[10] C. Slimings, T.V. Riley, Antibiotics and hospital-acquired Clostridium difficile -associated diarrhoea: a systematic review, J. Antimicrob. Chemother. 51 (2013) 1339e1350. [11] S. Dial, J.A.C. Delaney, V. Schneider, S. Suissa, Proton pump inhibitor use and risk of community-acquired Clostridium difficile -associated disease defined by prescription for oral vancomycin therapy, CMAJ 175 (2006) 745e748. [12] D. Bobak, L.M. Arfons, R.J. Creger, H.M. Lazarus, Clostridium difficile -associated disease in human stem cell transplant recipients: coming epidemic or false alarm? Bone Marrow Transpl. 42 (2008) 705e713. [13] J.F. Rodemann, E.R. Dubberke, K.A. Reske, H. Seo, C.D. Stone, Incidence of Clostridium difficile infection in inflammatory bowel disease, Clin. Gastroenterol. Hepatol. 5 (2007) 339e344. [14] E. Castagnola, T. Battaglia, R. Bandettini, I. Caviglia, I. Baldelli, M. Nantron, C. Moroni, A. Garaventa, Clostridium difficile -associated disease in children with solid tumors, Support Care Cancer 17 (2009) 321e324. [15] T.J. Sandora, M. Fung, K. Flaherty, L. Helsing, P. Scanlon, G. Potter-Bynoe, C.A. Gidengil, G.M. Lee, Epidemiology and risk factors for Clostridium difficile infection in children, Pediatr. Infect. Dis. J. 30 (2011) 580e584. [16] C.A. Muto, M. Pokrywka, K. Shutt, A.B. Mendelsohn, K. Nouri, K. Posey, T. Roberts, K. Croyle, S. Krystofiak, S. Patel-Brown, A.W. Pasculle, D.L. Paterson, M. Saul, L.H. Harrison, A large outbreak of Clostridium difficile-associated disease with an unexpected proportion of deaths and colectomies at a teaching hospital following increased fluoroquinolone use, Infect. Control Hosp. Epidemiol. 26 (2005) 273e280. [17] J. Freeman, J. Vernon, K. Morris, S. Nicholson, S. Todhunter, C. Longshaw, M.H. Wilcox, Pan-European Longitudinal Surveillance of Antibiotic Resistance among Prevalent Clostridium difficile Ribotypes' Study Group, Pan-European longitudinal surveillance of antibiotic resistance among prevalent Clostridium difficile ribotypes, Clin. Microbiol. Infect. 21 (2015) 248 e9-248.e16. pin, M.E. Alary, L. Valiquette, E. Raiche, J. Ruel, K. Fulop, D. Godin, [18] J. Pe C. Bourassa, Increasing risk of relapse after treatment of Clostridium difficile colitis in Quebec, Canada, Clin. Infect. Dis. 40 (2005) 1591e1597. ez, E. Cercenado, L. Alcal pez, J. Martínez[19] T. Pela a, M. Marín, A. Martín-Lo n, P. Catal Alarco an, M. S anchez-Somolinos, E. Bouza, Metronidazole resistance in Clostridium difficile is heterogeneous, J. Clin. Microbiol. 46 (2008) 3028e3032. [20] S.M. Trifilio, J. Pi, J. Mehta, Changing epidemiology of Clostridium difficileassociated disease during stem cell transplantation, Biol. Blood Marrow Transpl. 19 (2013) 405e409.
Please cite this article in press as: P. Spigaglia, et al., Clostridium difficile infection (CDI) in children due to hypervirulent strains PCR-ribotype 027: An emblematic report of two cases, Anaerobe (2015), http://dx.doi.org/10.1016/j.anaerobe.2015.09.004
Contents lists available at ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Clostridium difficile infection (CDI) in children due to hypervirulent strains PCR-ribotype 027: An emblematic report of two cases Patrizia Spigaglia a, *, Fabrizio Barbanti a, Elio Castagnola b, Roberto Bandettini c , Rome, Italy Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanita Infectious Diseases Unit, Istituto Giannina Gaslini, Genoa, Italy c Clinical Pathology Laboratory Unit, Istituto Giannina Gaslini, Genoa, Italy a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 July 2015 Received in revised form 10 September 2015 Accepted 15 September 2015 Available online xxx
In this report, the first two cases of pediatric Clostridium difficile infection (CDI) due to the hypervirulent PCR-ribotype 027 in Italy are described as emblematic of the role of both the infecting C. difficile strain and patient status in the occurrence and clinical manifestation of CDI in children. © 2015 Elsevier Ltd. All rights reserved.
Keywords: C. difficile Children PCR-ribotype 027 HSCT GVHD Antibiotics
1. Introduction An increased incidence of Clostridium difficile infection (CDI) has been recently observed in pediatric patients, particularly in North America where PCR-ribotype 027 (North American pulsed-field type 1, NAP1) has been recognized as cause of nosocomial diarrhea in 10e24% of symptomatic children [1,2]. Hypervirulent PCRribotype 027 has emerged in the last decade and it is now endemic throughout the U.S., Canada, and Europe [3]. C. difficile strains 027 show resistance to fluoroquinolones, production of binary toxin (CDT) and alterations of the tcdC gene, coding for the negative toxins regulator. This PCR-ribotype, associated with increased morbidity and mortality of infection in adults may be contributing to increasing pediatric CDI rates as well. In Italy, PCR-ribotype 027 is emerging and it actually represents the 9% of the toxigenic strains isolated in hospitals [4]. In this report, we described the first two cases of CDI by PCR-ribotype 027
* Corresponding author. Department of Infectious, Parasitic and ImmuneMediated Diseases, Istituto Superiore di Sanit a, Viale Regina Elena 299, 00161 Rome, Italy. E-mail address: [email protected] (P. Spigaglia).
in Italy as emblematic of the role of both the infecting C. difficile strain and patient status in the occurrence and clinical manifestation of infection.
2. Case one A 17-year-old female was hospitalized in October 2014. The patient was in immunosuppressive therapy for chronic graftversus-host disease (GVHD), following allogeneic hemopoietic stem cell transplant (allo-HSCT) for acute leukemia. The patient, with chronic lung disease due to GVHD and a Pseudomonas aeruginosa chronic infection, was undergoing to antibiotic treatment with piperacillin-tazobactam and tobramycin. She was treated empirically with ciprofloxacin because of a worsening of respiratory disease in another structure, located in a different Italian region, and then transferred in the Gaslini hospital. At time of admission, the patient presented abdominal pain and distension, diarrhea (5 lousy stools/day), temperature of 36 C, pulse rate of 68 beats/min, respiratory rate of 24/min, blood pressure of 125/83 mm Hg and oxygen saturation of 98%. Patient's clinical history reported an episode of C. difficile intestinal disease by a toxigenic strain in December 2012. In three following hospitalizations in 2013, the
http://dx.doi.org/10.1016/j.anaerobe.2015.09.004 1075-9964/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: P. Spigaglia, et al., Clostridium difficile infection (CDI) in children due to hypervirulent strains PCR-ribotype 027: An emblematic report of two cases, Anaerobe (2015), http://dx.doi.org/10.1016/j.anaerobe.2015.09.004
2
P. Spigaglia et al. / Anaerobe xxx (2015) 1e3
screening for C. difficile was always negative. After admission in our hospital, laboratory analysis revealed a white blood cell count (WBC) of 6.9 103/ml with 92.8% of neutrophils, fecal samples were negative for Adenovirus, Rotavirus, Cryptosporidium and Helicobacter pylori tests but positive for presumptive C. difficile PCRribotype 027 using the Xpert® C. difficile (Cepheid), according to manufacturer guidelines. The patient was treated for CDI with vancomycin (125 mg/6 h) for ten days. The clinical picture was further complicated for the development of a gastrointestinal tract graft-versus-host disease (GI GVDH) with presence of diarrhea (up to 7 lousy stools/day) and absence of fever. The patient was discharged at the 13th day of hospitalization. 3. Case two A 7-year-old male patient was hospitalized in October 2014. The patient was previously admitted in another hospital of the same Italian region and, after two days, he was transferred in the Gaslini hospital for abdominal pain. No clinical history of previous underlying disease. At time of admission, the patient presented a clinical picture of acute abdomen, temperature of 36 C, pulse rate of 102 beats/min, respiratory rate of 22/min and blood pressure of 121/ 73 mm Hg. An initial diagnosis of acute appendicitis was made and ampicillin-sulbactam and metronidazole were administered as therapy. The day after admission, the patient underwent surgery for acute appendicitis. The day after surgery, the patient presented a mild diarrhea (2 lousy stools in 12 h). Laboratory analysis revealed a WBC of 10.7 103/ml with a differential of 77.8% of neutrophils and C-RP of 9.18 mg/dl. Fecal cultures were negative for Salmonella spp, Shigella spp and Campylobacter spp. but positive for presumptive C. difficile PCR-ribotype 027 using the Xpert® C. difficile (Cepheid), according to manufacturer guidelines. Fecal sample was negative to Rotavirus, Adenovirus and Norovirus tests. The patient was treated with metronidazole (30e40 mg/kg/die) and vancomycin (40 mg/ kg/die). The body temperature was always around 36 C, except on the third day after surgery (37.9 C). Three days after the beginning of treatment the clinical picture improved (WBC of 10 103/ml, 70.5% of neutrophils and C-RP of 1.47 mg/dl) and the patient had formed stool on the fourth day. The patient was discharged from the hospital 11 days after the admission, in good clinical conditions (temperature of 36.4 C, WBC of 10.3 103/ml, 64.2% of neutrophils and C-RP < 0.46 mg/dl). 4. Detection, typing and characterization of strains In both cases, fecal samples were inoculated on Brucella Blood Agar and plates were incubated for 48 h at 37 C in Bio-Bag™ Environmental Chambers (Becton Dickinson) for anaerobic bacteria culture. Culture plates were sent to the Istituto Superiore di Sanita for C. difficile typing and analysis of antibiotic susceptibility. Briefly, C. difficile strains were isolated on the selective plates chromID™ rieux) after 48 h of incubation at 35 C under C. difficile (bioMe anaerobic conditions (85% N2, 10% H2 and 5% CO2). Results obtained by Xpert® C. difficile (Cepheid) were confirmed by C. difficile typing using the capillary gel electrophoresis PCR-ribotyping method, as described by Indra et al. [5]. PCR-ribotypes were determined by submitting data to the free web-database (http://webribo.ages.at). The minimum inhibitory concentrations (MICs) for moxifloxacin (MXF), erythromycin (ERY), clindamycin (CLI), rifampicin (RIF), metronidazole (MTZ) and vancomycin (VAN) were determined by Agar Dilution method on Brucella agar plates containing 1 mg/L vitamin K, 5 mg/L haemin and 5% defibrinated sheep red blood cells. In accordance with the Clinical and Laboratory Standards Institute (CLSI) interpretative categories approved for anaerobic bacteria the breakpoints for MXF and CLI was 8 mg/L, whereas the
breakpoint for MTZ was 32 mg/L [6]. The breakpoints for RIF, ERY and VAN were 4, 8 and 16 mg/L, respectively, in accordance with the CLSI interpretive categories approved for Staphylococcus aureus [6], since no values were provided for anaerobes. 5. Discussion The first two pediatric cases of CDI due to PCR-ribotype 027 in Italy occurred in a pediatric hospital located in the north of the Country. Both patients were transferred in the hospital from other structures. No epidemiologic evidence of patient-to-patient transmission of the same strain was found, and no outbreaks or cases of diarrhea occurred during the period in which the two patients were recovered. Unfortunately, no C. difficile epidemiological data are available about the hospitals in which the patients were firstly admitted. In this study, both patients did not show clinically signs and symptoms compatible with a severe CDI, as defined by the European Society of Clinical Microbiology and Infectious Diseases [7], even if infected by a hypervirulent strain. It has been observed that C. difficile PCR-ribotype does not always can be considered predictive of severe disease [8]. Furthermore, a recent study performed in U.S.A. indicate few markers of severe disease of CDI in HSCT pediatric patients, also if infected by strains 027 [9], probably because immunosuppression may attenuate the disease. Several risk factors for the development of CDI are known, first of all medication administration (e.g., antibiotics, acid suppression medication, and chemotherapy) [10,11]. In adults, inflammatory bowel disease (IBD) and human stem cell transplantation have been associated with CDI [12,13], whereas in children solid tumors, organ transplantation, gastrostomy, or jejunostomy represent a further risk for this infection [14,15]. In the cases here described, both patients were subject to antibiotics therapy before developing CDI: patient of case one was treated with ciprofloxacin due to a chronic lung GVHD and a P. aeruginosa infection, while patient of case two has a perioperative treatment with ampicillin-sulbactam plus metronidazole. As known, fluoroquinolones have been significantly associated to CDI due to strains 027 [16]. Both strains analyzed were multi-drug resistant (MDR) with high MIC to MXF (32 mg/L), ERY (MIC 256 mg/L), RIF (MIC 32 mg/L) and MIC ¼ 8 mg/L to CLI. Both strains were susceptible to MTZ and VAN, with MIC ¼ 0.25 mg/ L for both antibiotics. In recent papers, high geometric mean of MICs to MTZ of strains 027 [17] and cases of treatment failures of CDI cause by PCR-ribotype 027 after therapy with MTZ have been described [18]. In this study, analysis of antibiotic susceptibility was performed after freezing and several passages on culture plates that can cause MIC values reduction to MTZ, as previously observed [19]. Interestingly, when strains were subjected to sub-concentrations of MTZ, some colonies reached a MIC ¼ 8 mg/L (data not shown). Therefore, the capability to survive in vivo in presence of multiple antibiotics, including MTZ, play a key role in the occurrence of CDI independently from the status of the patient. This report describe two emblematic cases of CDI due to PCRribotype 027 in children. Clinical manifestations of CDI in these patients was not severe, despite the characteristics of hypervirulence of the infecting C. difficile strains. Anyway, strain characteristics have probably a crucial role in survival of the pathogen and occurrence of infection. Pediatric population subgroups, including HSCT patients and those who received multiple classes of antibiotics, are more susceptible to CDI. HSCT children seem to be particularly prone to CDI in the first year after transplantation, as observed in adults [20]. These patients can undergo to CDI recurrences and an association between CDI and GI GVDH seems to be possible. All these features should be considered with particular
Please cite this article in press as: P. Spigaglia, et al., Clostridium difficile infection (CDI) in children due to hypervirulent strains PCR-ribotype 027: An emblematic report of two cases, Anaerobe (2015), http://dx.doi.org/10.1016/j.anaerobe.2015.09.004
P. Spigaglia et al. / Anaerobe xxx (2015) 1e3
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Please cite this article in press as: P. Spigaglia, et al., Clostridium difficile infection (CDI) in children due to hypervirulent strains PCR-ribotype 027: An emblematic report of two cases, Anaerobe (2015), http://dx.doi.org/10.1016/j.anaerobe.2015.09.004