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Shigellosis and toxic megacolon secondary to Shigella flexneri serotype 3a: The challenges of laboratory diagnosis

Open AccessPublished:March 01, 2018DOI:https://doi.org/10.1016/j.ijid.2018.02.020

      Highlights

      • Pitfalls of molecular platforms illustrated.
      • Rare but life-threatening complications demonstrated in immunocompetent adult.
      • Azithromycin resistance within endemic Shigella outbreaks among MSM in the UK discussed.

      Abstract

      We present a rare case of Shigella flexneri bacteraemia and toxic megacolon, and discuss the challenges of conventional laboratory techniques versus molecular PCR platforms in differentiating between Shigella species and Escherichia coli.

      Keywords

      Background

      Shigella is a leading cause of diarrhoea globally, causing approximately 80–165 million cases per year (
      • Bowen A.
      Infectious diseases related to travel.
      ). It is a pathovar of Escherichia coli; with the primary infectious species worldwide being Shigella flexneri. Laboratory detection of Shigella species typically involves isolation on selective media, followed by biochemical tests, such as indole and lysine decarboxylase, to differentiate between Shigella and E. coli. Species identification is then confirmed by serotyping of the surface antigens. With the advent of molecular testing, including PCR, accurate detection and speciation of Shigella has increased. Time to detection has reduced to hours compared to conventional methods which can take days. Implementation of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) within diagnostic microbiology laboratories has reduced this time even further, to just a few minutes, but comes at the cost of difficulty in distinguishing between closely related species (
      • Deng J.
      • Fu L.
      • Wang R.
      • Yu N.
      • Ding X.
      • Jiang L.
      • et al.
      Comparison of MALDI-TOF MS, gene sequencing and the Vitek 2 for identification of seventy-three clinical isolates of enteropathogens.
      ). Here we present a case of S. flexneri 3a shigellosis, bacteraemia and toxic megacolon, whose diagnosis was hampered and then resolved using two molecular platforms.

      Case description

      A 44-year-old British man was admitted with a 3-week history of diarrhoea, passing liquid stools five to six times per day with no blood or mucus passed per rectum. He had lost six kilograms in weight and had developed nearly constant hiccupping. There was no significant past medical history, nor had he travelled outside the UK for several years. The patient had a single long-term male sexual partner with no reported recent high-risk sexual contact.
      On clinical examination, there was marked dehydration but no focal signs. He was tachycardic (114 bpm) and hypothermic (34.8 °C), but the remainder of his observations were normal. Blood tests on admission revealed a neutrophilia (15.9 × 109/L) with an otherwise unremarkable full blood count. He was hyponatraemic (112 mmol/L), and the CRP was elevated (183 mg/L).
      The clinical impression was of gastroenteritis with hyponatraemia secondary to profound dehydration. However, an urgent CT scan of his abdomen demonstrated circumferential bowel wall thickening involving the terminal ileum, entire colon, and rectum with surrounding stranding. There was marked dilatation of the transverse colon up to 7.9 cm in diameter consistent with toxic megacolon (see Fig. 1).
      Fig. 1
      Fig. 1CT abdomen and pelvis with contrast on day 6 of admission demonstrating new large volume pneumoperitoneum and a moderate volume of free intraperitoneal fluid consistent with recent perforation. The site of perforation is presumed to be the grossly dilated transverse colon which measured 9.4 cm in maximal diameter.
      The surgical team advised no immediate intervention, but suggested that the admitting gastroenterology team conduct daily abdominal radiographs. Intensive care and endocrinology consults were conducted, and these teams followed him closely while his hyponatraemia resolved with rehydration. Regular IV piperacillin/tazobactam and IV amikacin were commenced, as well as IV metronidazole and PO vancomycin to cover for the possibility of Clostridium difficile colitis. Azithromycin was later added for empirical treatment of possible Salmonella or Shigella infection.
      After the microbiological diagnosis was confirmed the patient was reviewed. He appeared stable, with resolution of his diarrhoea and no episodes of fever. However, progressive dilatation of the transverse colon to 17 cm in maximum diameter was demonstrated on serial abdominal radiographs.
      On day 6 of admission, before a planned rationalization of treatment to IV ciprofloxacin was instituted, the patient developed severe generalized abdominal pain. A CT scan of his abdomen revealed extensive pneumoperitoneum and new intraperitoneal fluid consistent with recent perforation. An emergency laparotomy with sub-total colectomy was performed with formation of an end ileostomy. Intra-operative and histological findings were consistent with florid pan-colitis with ulceration and perforation. The patient had an uneventful post-operative recovery, completed a 10-day course of IV ciprofloxacin, and was discharged with plans for colorectal surgical follow-up.

      Laboratory detection

      On day 2 of admission, a Gram-negative blood culture isolate was identified by Bruker MALDI Biotyper (Bruker Daltonics, Leipzig, Germany, Version 3) as E. coli/Shigella species with a score of 2.3. The organism was noted to be a non-lactose fermenter, but given the prevalence of E. coli bacteraemia in the UK, this was thought the most likely aetiological agent, despite the initial presentation of gastroenteritis. Antibiotic sensitivity testing by disc diffusion (BSAC methods) demonstrated susceptibility to piperacillin/tazobactam, amikacin and ciprofloxacin, which was confirmed by the Microscan WalkAway system (Siemens Healthcare Diagnostics, Deerfield, Il, USA). Additional sensitivity testing to azithromycin demonstrated a MIC of 1.0 mg/L. HIV serology was negative.
      In light of the clinical picture of toxic megacolon, the sexual history and consideration that only 10% of E. coli are non-lactose fermenters, the MALDI-TOF result was reviewed. As the stool culture was negative for Salmonella species, Shigella species, Campylobacter species and E. coli 0157, the stool specimen was then screened using the BD MAX®™ Enteric Bacterial Panel (Becton, Dickinson and Company; Sparks, Maryland, USA). Within 3 h, the PCR platform detected Shigella species/Enteroinvasive E. coli DNA and subsequent serological agglutination confirmed the blood culture isolate was S. flexneri serotype 1–6. An API20E performed on the blood culture isolate was consistent with a Shigella species (API20E 0004100), albeit with a low discriminatory value (69%).
      The Shigella isolate was referred to PHE where it was identified as S. flexneri serotype 3a. Whole genome sequencing (WGS) was performed and the single nucleotide polymorphism (SNP) address of this isolate was compared to that of other UK isolates. Although no identical organisms were referred to PHE, this organism was found to be part of a wider cluster of 70 cases in the UK. OXA-1 and TEM-1 genes which encode for beta-lactamases and a mutation in the mdf(A) gene associated with azithromycin resistance were identified by WGS in this isolate.

      Discussion

      Although exceedingly rare in immunocompetent adults, Shigella should be considered as a cause of toxic dilatation. UK cases are usually travel-related. However, sexual transmission among men who have sex with men (MSM) is well documented and increasingly prevalent, with Public Health England (PHE) investigating an on-going national outbreak of S. flexneri since 2011 (
      • Baker K.
      • Dallman T.
      • Ashton P.
      • Day M.
      • Hughes G.
      • Crook P.
      • et al.
      Intercontinental dissemination of azithromycin-resistant shigellosis through sexual transmission: a cross-sectional study.
      ). This case highlights the need for thorough microbiological testing and interpretation, as well as a high index of suspicion for Shigellosis, especially as such patients are often considered for steroids or other immunosuppressive treatment for presumed inflammatory bowel disease.
      The laboratory diagnostics employed in this case are associated with a number of potential caveats that must be carefully considered before use and when interpreting results. While MALDI-TOF has revolutionised bacterial identification in clinical microbiology laboratories with its speed and accuracy, this case highlights the importance of laboratory staff having access to sufficient clinical details to correctly interpret the result. This is especially so with closely related species, such as E. coli and Shigella species (
      • Deng J.
      • Fu L.
      • Wang R.
      • Yu N.
      • Ding X.
      • Jiang L.
      • et al.
      Comparison of MALDI-TOF MS, gene sequencing and the Vitek 2 for identification of seventy-three clinical isolates of enteropathogens.
      ). With these two organisms, serotyping remains a valuable tool despite the need for a pure culture and staff expertise.
      Stool PCR platforms, which are now able to detect most common diarrhoeal pathogens, afford obvious advantages in high throughput testing. In this case, the BD MAX platform was able to detect Shigella species in the culture-negative stool, highlighting the ability of molecular platforms to detect certain species at a lower limit of detection compared to routine faecal culture (
      • Deng J.
      • Fu L.
      • Wang R.
      • Yu N.
      • Ding X.
      • Jiang L.
      • et al.
      Comparison of MALDI-TOF MS, gene sequencing and the Vitek 2 for identification of seventy-three clinical isolates of enteropathogens.
      ).
      Azithromycin resistance is increasingly reported among Shigella species in MSM outbreaks worldwide (
      • Baker K.
      • Dallman T.
      • Ashton P.
      • Day M.
      • Hughes G.
      • Crook P.
      • et al.
      Intercontinental dissemination of azithromycin-resistant shigellosis through sexual transmission: a cross-sectional study.
      ). However, assessing azithromycin susceptibility in Shigella infection is also problematic, with an absence of clinically accepted guidelines for antimicrobial susceptibility testing (AST). Our isolated demonstrated an MIC of 1.0 mg/L, but given that blood levels of azithromycin rarely exceed 1 mg/L it is unclear whether an earlier switch to ciprofloxacin would have prevented GI perforation. WGS, though not an approved method of AST, is beginning to be used as a novel tool for detecting antibiotic resistance in clinical isolates (
      • Ellington M.
      • Ekelund Q.
      • et al.
      The role of whole genome sequencing in antimicrobial susceptibility testing of bacteria: report from the EUCAST subcommittee.
      ). In this case WGS identified a mutation within the chromosomal gene mdf(A), which has been associated with azithromycin resistance among Shigella species, however it is unclear whether this mutation directly infers phenotypic resistance (
      • Baker K.
      • Dallman T.
      • Ashton P.
      • Day M.
      • Hughes G.
      • Crook P.
      • et al.
      Intercontinental dissemination of azithromycin-resistant shigellosis through sexual transmission: a cross-sectional study.
      ,
      • Edgar R.
      • Bibi E.
      MdfA, an Escherichia coli multidrug resistance protein with extraordinary broad spectrum of drug recognition.
      ).
      Our case highlights the evolving role of molecular diagnostics in identifying enteric pathogens, which can be difficult to culture, while demonstrating the important role that traditional techniques have in those rare instances where molecular platforms lack sensitivity.

      Declarations

      The costs of the Bruker MALDI-TOF and the BD MAX testing were supported by grants from Barts and the London Charity . We declare no competing interests.

      Acknowledgements

      We thank Dr Claire Jenkins at the Gastrointestinal Bacteria Reference Unit (GBRU) at PHE and Professor Neil Woodford at the Antimicrobial Resistance and Healthcare-Associated Infections (AMRHAI) Reference Unit at PHE for their guidance in interpretation of WGS in this case.

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