Advertisement

Toxinotype A Clostridium perfringens causing septicaemia with intravascular haemolysis: two cases and review of the literature

Open AccessPublished:December 17, 2021DOI:https://doi.org/10.1016/j.ijid.2021.12.331

      Highlights

      • Clostridium perfringens infection with intravascular haemolysis is rare.
      • Mortality remains 80%.
      • In the literature, polymerase chain reaction toxinotyping is described in only four cases: all toxinotype A.
      • Toxinotyping is important for insight into pathophysiology and treatment development.

      ABSTRACT

      Background

      Septicaemia with intravascular haemolysis is a rare, but often fatal, presentation of Clostridium perfringens infection. C. perfringens is a Gram-positive, anaerobic bacterium that can produce multiple toxins. Toxinotyping is not performed regularly.

      Methods

      This article describes two human cases of C. perfringens infections. Toxinotyping was performed using polymerase chain reaction (PCR). Additionally, a structured review of the literature was performed which searched specifically for cases of C. perfringens infection with haemolysis.

      Results

      Both cases were identified as toxinotype A strains and both cases were fatal. Also, both cases showed marked haemolysis during their clinical course, which is assumed to have played a significant role in their outcome. In total, 83 references were identified describing human C. perfringens infection with haemolysis. Mortality rates have been stable over the last 10 years at 80%. Toxinotyping has been performed in a total of six cases. Of the four cases analysed by PCR, all were identified as toxinotype A.

      Conclusions

      Haemolytic C. perfringens infections are rare but are fatal in most cases. Toxinotyping is performed rarely. The authors advocate increased use of toxinotyping to gain insight into pathophysiology and more effective interventions.

      Keywords

      Introduction

      Clostridium perfringens (C. perfringens) is a Gram-positive, anaerobic, rod-shaped bacterium which is commonly found in soil, food and intestinal tracts of humans and animals (
      • Kiu R
      • Hall LJ.
      An update on the human and animal enteric pathogen Clostridium perfringens.
      ). C. perfringens infections show a broad spectrum of clinical manifestations, including intestinal infections by specific toxinotypes, asymptomatic bacteraemia, gangrene, massive intravascular haemolysis and multi-organ failure from septic shock. Septicaemia with subsequent haemolysis is notorious for rapid clinical deterioration and death (80% mortality) (
      • van Bunderen CC
      • Bomers MK
      • Wesdorp E
      • Peerbooms P
      • Veenstra J.
      Clostridium perfringens septicaemia with massive intravascular haemolysis: a case report and review of the literature.
      ).
      C. perfringens can produce a wide array of more than 20 toxins (
      • Kiu R
      • Hall LJ.
      An update on the human and animal enteric pathogen Clostridium perfringens.
      ). The combination of six of these toxins [alpha toxin, beta toxin, epsilon toxin, iota toxin, C. perfringens enterotoxin and necrotic enteritis B-like toxin] is used to determine the toxinotype (A to G) (
      • Rood JI
      • Adams V
      • Lacey J
      • Lyras D
      • McClane BA
      • Melville SB
      • et al.
      Expansion of the Clostridium perfringens toxin-based typing scheme.
      ). Based on animal and in-vitro studies, the alpha toxin appears to be the major virulence factor in human C. perfringens septicaemia with gas gangrene and haemolysis (
      • Flores-Díaz M
      • Alape-Girón A.
      Role of Clostridium perfringens phospholipase C in the pathogenesis of gas gangrene.
      ;
      • Stevens DL
      • Aldape MJ
      • Bryant AE.
      Life-threatening clostridial infections.
      ;
      • Kiu R
      • Hall LJ.
      An update on the human and animal enteric pathogen Clostridium perfringens.
      ). However, it is likely that other toxins, such as theta toxins, contribute in disease development (
      • Stevens DL
      • Bryant AE.
      Role of clostridial toxins in the pathogenesis of gas gangrene.
      ;
      • Suzaki A
      • Ohtani K
      • Komine-Aizawa S
      • Matsumoto A
      • Kamiya S
      • Hayakawa S.
      Pathogenic characterization of Clostridium perfringens strains isolated from patients with massive intravascular hemolysis.
      ).
      This article describes two fatal cases of toxinotype A C. perfringens septicaemia causing massive intravascular haemolysis. In addition, an overview of all published literature (since 1990) from human cases is provided.

      Case 1

      A 65-year-old male with a history of type 2 diabetes, subclinical hypothyroidism and ischaemic stroke was admitted to the Emergency Department (ED) with a 2-day history of acute abdominal pain. Over the previous 3 weeks, symptoms had included loss of appetite, confusion, vague upper stomach pain and general malaise. On arrival, he was afebrile with an alert mental status and severe abdominal pain. Vital signs were unremarkable, except for tachypnoea (25–30 breaths/min). Physical examination revealed severe diffuse abdominal pain showing signs of peritoneal irritation. Computed tomography (CT) scan showed extensive gas-forming liver abscesses with subcapsular gas, aerobilia with signs of intraperitoneal breakthrough. Broad-spectrum antibiotics (cefuroxime, metronidazole and single-dose tobramycin) were administered, and two hepatic drains were placed (CT-guided). Within the first hour of placement of the drains, 3 L of sanguinolent fluid was drained. Subsequently, the patient developed septic shock with jaundice and haemoglobinuria. Laboratory findings revealed a significant decrease in haemoglobin levels (from 6.8 mmol/L to 0.9 mmol/L within a 6-h interval) caused by massive intravascular haemolysis with metabolic acidosis (pH 6.93, bicarbonate 7 mmol/L, lactic acid 14.9 mmol/L). This raised the suspicion of C. perfringens infection, and penicillin and ciprofloxacin (for possible mixed bacterial flora) were added to the antibiotic regime.
      The next day, blood and hepatic fluid cultures revealed C. perfringens, and the patient was transferred to an academic centre for further management and laparotomy. A necrotic gall bladder and 3 L of abdominal blood were removed. Gangrenous cholecystitis was the source of infection. Despite further antibiotic escalation, the patient died within hours of surgery due to refractory sepsis and multi-organ failure.

      Case 2

      A 69-year-old male with a history of chronic obstructive pulmonary disease, hypertension and gastrointestinal stromal tumour with liver metastases presented to the ED with a 2-day history of nausea, vomiting, dyspnoea and increasing jaundice. The gastrointestinal stromal tumour had been treated with avapritinib for 6 months with good response. The primary tumour had been resected approximately 15 months prior to presentation, and four remaining liver metastases had been treated with percutaneous microwave ablation 12 days earlier.
      On presentation, the patient was tachypnoeic (30 breaths/min) with 88% SpO2, tachycardic (122 beats/min), and jaundiced with mottling and a prolonged capillary refill time. Blood pressure was 142/82 mmHg. The right upper abdominal quadrant was tender. Ultrasonography showed no evident liver abscess(es), but hyperechoic speckles in the hepatic veins were observed, suspected to be gas bubbles. Oxygen supplementation, fluid resuscitation and broad-spectrum antibiotics (cefuroxime, gentamicin and metronidazole) were initiated.
      Within 1 h of presentation, the patient deteriorated acutely, with decreased consciousness and progressive respiratory failure. This was followed by circulatory arrest due to intermittent asystole and pulseless electric activity. Blood chemistry showed increasing lactic acidosis (16 mmol/L), decreasing haemoglobin level (from 6.5 to 2.1 mmol/L in 2 h despite transfusion) and progressive hyperkalaemia (from 4.9 to 9.0 mmol/L despite treatment). After 2.5 h of cardiopulmonary resuscitation, continuing resuscitation was deemed futile, and the patient died. Additional blood results showed intravascular haemolysis (free haemoglobin 317 µmol/L and hyperbilirubinaemia of 264 µmol/L), and blood cultures grew C. perfringens, Clostridium ramosum and Bacteroides fragilis.

      Methods

      Literature review

      PubMed was searched on 7 January 2021: ((("haemolysis" [tiab]) OR ("hemolysis" [tiab])) AND ("clostridium perfringens" [tiab])), period 1990–2020. All articles in the English language reporting on human cases (age ≥16 years) with a C. perfringens infection and intravascular haemolysis were included.

      Toxinotyping

      Polymerase chain reaction (PCR) toxinotyping was performed according to the method described by
      • Rood JI
      • Adams V
      • Lacey J
      • Lyras D
      • McClane BA
      • Melville SB
      • et al.
      Expansion of the Clostridium perfringens toxin-based typing scheme.
      .

      Results

      The PubMed search returned 138 results, of which 81 references described 83 cases of human C. perfringens infection with intravascular haemolysis (Table 1). The mortality rate in published cases appears to have remained unchanged over the past 10 years (30/38: 79%). In two cases, a non-PCR method was used to detect alpha toxin, while in four cases (5%), PCR toxinotyping on various toxins was performed. In the cases analysed by PCR, only the alpha-toxin gene was detected (i.e. toxinotype A). Multiplex PCR toxinotyping of the isolates of Cases 1 and 2 classified both as toxinotype A.
      Table 1Literature overview of human cases with Clostridium perfringens infection and haemolysis.
      AuthorYearAgeSexSurvivalOrigin of infectionPositive cultureToxin typingMethod of typingToxinotype
      1Bätge199261MYesLiver abscessBloodNR--
      2Ifthikaruddin199254FNoUnknownBloodNR--
      3Hübl199384FNoUnknown (intestinal?)Blood (i)α toxin (iii)Turbidimetric assay (serum PLC activity)-
      4Rogstad199361MNoLiver abscess (micro)Blood, liver, spleenNR--
      5Clarke199453FYesNecrotizing enteritisBlood, peritoneal fluidNR--
      6Gutiérrez199574MNoLiver abscess (micro)BloodNR--
      7Meyerhoff199566FNoUnknownBloodNR--
      8Bush199658FYesUnknown (post-laparoscopic cholecystectomy)BloodNR--
      9Jones199666FNoLiver abscessBlood, liver abcsessNR--
      10Pun199647MNoCholangitisBlood (i)NR--
      11Singh199673FNoUnknownBloodNR--
      12Singer199755FNoUnknownBloodNR--
      13Alvarez199977FNoAbdominalBloodNR--
      14Thomas199973MYesCholecystitisBlood (i)NR--
      15Eckel200065FYesLiver abscessBlood (i)NR--
      16Barrett2002NRFNoSeptic spontaneous abortionBloodNR--
      17Halpin200229FYesPostcaesarean endometritisBloodNR--
      18Hamoda200239FYesPostamniocentesis endometritisBloodNR--
      19Jimenez200279MNoUnknownBloodNR--
      20Kreidl200280MNoLiver abscessBlood, liver abcsessNR--
      21Ikegami200467MYesAcute pancreatitisPancreasNR--
      22Solis200450MNoHepatic gas gangrene + mycotic aneurysm of dCHADonor liver and hepatic arteryNR--
      23Vaiopoulos200474MNoIntestinal and biliaryBloodNR--
      24Au200565MNoLiver abscessNRNR--
      25Pirrotta200550MNoUnknownBlood and stoolNR--
      26Rodriguez200557MNoBiliaryBloodNR--
      27Daly200680MNoLiver abscessBloodNR--
      28Eigneberger200660MNoLiver abcsessLiver (gram staining)NR--
      29Kwon200671FNoUnknownBlood (i)NR--
      30Loran200669FNoLiver abscessNRNR--
      31McArthur200649MNoAbdominalBloodNR--
      32Ohtani200678MNoLiver abscessBlood, liver abcsessNR--
      33Kapoor200758MNoUnknownBloodNR--
      34Poon200764FNoUnknown (hepatobiliary?)Blood (i)NR--
      35Poulou200774MNoUnknownBloodLecithinase (iii)Turbidity on the egg yolk medium-
      36Egyed200839FYesUnknownBloodNR--
      37Hess200881MNoAcute diverticulitisBlood, brain, heart, spleen (i)NR--
      38Nadisauskiene200831FNoPostcaesarean endometritisBloodNR--
      39Boyd200946MNoAcalculous cholecystitisBloodNR--
      40Uppal200961MNoUnknownBloodNR--
      41Bryant201060FYesUterusBlood and intrauterine samplesNR--
      42Bunderen201074MYesCholangitisBloodNR--
      43Merino201083FNoLiver abscessBloodNR--
      44Ng201061FYesLiver abscessBlood (i)NR--
      45Rajendran201058MYesLiver abscessBlood, liver abscess, gall bladderNR--
      46Stroumsa201141FYesInfected uterine myomaBlood (i)NR--
      47Law201250FNoLiver abscessBloodNR--
      48Qandeel201259MYesLiver abscess (post-laparoscopic cholecystectomy)BloodNR--
      49Watt201252MYesPan-enteritisBloodNR--
      50Cécilia201364MNoUnknownBloodNR--
      51Dutton201366MNoNRBloodNR--
      52Okon201371MNoUnknownBlood, CSF (i)NR--
      53Kitterer201471MNoLiver abscessBloodNR--
      54Kurasawa201465MNoLiver abscessBloodNR--
      55Renaudon-Smith201437MYesLiver abscessBloodNR--
      56Simon201479FNoUnknownBloodNR--
      57Cochrane201565FYesEmphysematous cholecystitisBloodNR--
      58Khan201577MNoCholecystitis/Liver abscessNo (ii)NR--
      59Li201571MYesLiver abscess (post-TACE)BloodNR--
      60Shindo201573FNoLiver abcsessLiver abscess (i)α toxinMultiplex PCR (iv)A
      61Yamaguchi201580-89FNoUnknownBile, pleural effusions (i)NR--
      62Carretero201665MYesLiver abscessBlood, liver abscess (i)NR--
      63Hashiba201682MNoLiver abscess, emphysematous cholecystitisBloodα toxinMultiplex PCR (v)A
      64Lim201658MNoLiver abscessBloodNR--
      65Medrano-Juarez201632MYesUnknownBlood (i)NR--
      66Sarvari201676FNoEmphysematous gastritisIntestinal and subcutaneous tissueNR--
      67Balan201771FNoUnknownBloodNR--
      68Ewing201753FNoNecrotizing fasciitis right armWoundNR--
      69Kent201774FNoEnteritis necroticans (based on symptoms)BloodNR--
      70Kukul201717MNoGastrointestinal tractQuadratus muscleNR--
      71Gelonch201866MNoLiver abscessNRNR--
      72Gelonch201863MNoLiver abscessNRNR--
      73Shibazaki201868FNoLiver abscessBloodNR--
      74Wild201881FNoUnknownBloodα toxinPCR (iv)A
      75Sakaue201976MNoLiver abscessBloodα toxinMultiplex PCR (vi)A
      76Uojima201983MNoLiver abscess (post-TACE)Liver abscessNR--
      77Chinen202080FNoLiver abscessBlood, liver abscess (i)NR--
      78Fujikawa202077FNoLiver abscessBloodNR--
      79Kawakami202083MNoPelvic abscessBlood, intra-abdominal samplesNR--
      80Koubaissi202050MNoAbdominal?BloodNR--
      81Olds202085FNoLiver abscessBloodNR--
      82Smit202061MNoLiver abscessBloodNR--
      83Smit202071FNoUnknownBlood (i)NR--
      84Woittiez202165MNoGangrenous cholecystitisBlood, liver abscessα toxinMultiplex PCR (vii)A
      85Woittiez202169MNoHepatogenic?Blood (i)α toxinMultiplex PCR (vii)A
      M, male; F, female; positive culture, positive for C. perfringens; post-TACE, after transarterial chemo-embolization because of hepatocellular carcinoma; dCHA, donor common hepatic artery; NR, not reported; CSF, cerebrospinal fluid; PLC, phospholipase C; PCR, polymerase chain reaction; (i) multi-microbial, including C. perfringens; (ii) Gram-positive anaerobic rods in liver parenchyma; (iii) only alpha toxin/lecithinase was analysed, no other typing toxins; (iv) alpha toxin, beta toxin, epsilon toxin, iota toxin and C. perfringens enterotoxin were analysed; (v) as for (iv), plus beta2-toxin; (vi) as for (iv) plus necrotic enteritis B-like toxin and binary enterotoxin of C. perfringens; (vii) as for (iv) plus necrotic enteritis B-like toxin.

      Discussion

      Rapid progression to septicaemia and massive intravascular haemolysis is a well-known but rare clinical presentation of C. perfringens infection. Intravascular haemolysis is attributed to secretion of alpha toxin and occurs in 7–15% of cases with bacteraemia (
      • van Bunderen CC
      • Bomers MK
      • Wesdorp E
      • Peerbooms P
      • Veenstra J.
      Clostridium perfringens septicaemia with massive intravascular haemolysis: a case report and review of the literature.
      ). The approximate doubling time of C. perfringens of 7 min and rapid upregulation of toxin production contribute to rapid patient deterioration (
      • McArthur HL
      • Dalal BI
      • Kollmannsberger C.
      Intravascular hemolysis as a complication of Clostridium perfringens sepsis.
      ). Median time between admission and death is 8 h (
      • van Bunderen CC
      • Bomers MK
      • Wesdorp E
      • Peerbooms P
      • Veenstra J.
      Clostridium perfringens septicaemia with massive intravascular haemolysis: a case report and review of the literature.
      ). Immediate appropriate antibiotic treatment and surgical source control are paramount: interventions before onset of massive haemolysis seem to improve survival (
      • Simon TG
      • Bradley J
      • Jones A
      • Carino G.
      Massive intravascular hemolysis from Clostridium perfringens septicemia: a review.
      ). In vitro, C. perfringens alpha-toxin production and activity can be suppressed within 15–45 min by either clindamycin, metronidazole or rifampin (
      • Stevens DL
      • Maier KA
      • Mitten JE.
      Effect of antibiotics on toxin production and viability of Clostridium perfringens.
      ). Cornerstones of treatment remain identical to sepsis treatment, namely early recognition and intervention with the specific aim of reducing toxin production (
      • Watt J
      • Amini A
      • Mosier J
      • Gustafson M
      • Wynne JL
      • Friese R
      • et al.
      Treatment of severe hemolytic anemia caused by Clostridium perfringens sepsis in a liver transplant recipient.
      ).
      The literature reveals a paucity of toxinotyping data in human cases. Including the strains included in this study, six strains have been PCR-typed and all were positive for alpha toxin alone. Alpha toxin is a membrane-disrupting protein which causes cell necrosis by hydrolysing phospholipids in the cell membrane (
      • Kiu R
      • Hall LJ.
      An update on the human and animal enteric pathogen Clostridium perfringens.
      ). Likewise, it causes spherocytosis and haemolysis of erythrocytes (
      • van Bunderen CC
      • Bomers MK
      • Wesdorp E
      • Peerbooms P
      • Veenstra J.
      Clostridium perfringens septicaemia with massive intravascular haemolysis: a case report and review of the literature.
      ). Furthermore, alpha toxin decreases cardiac contractility, inhibits trafficking of immune cells such as neutrophils to the site of infection, and causes a micro-aerophilic environment by local coagulation and vasoconstriction, all favouring C. perfringens overgrowth (
      • Kiu R
      • Hall LJ.
      An update on the human and animal enteric pathogen Clostridium perfringens.
      ). All C. perfringens toxinotypes should, in theory, be able to induce intravascular haemolysis, as all types can produce alpha toxin (
      • Kiu R
      • Hall LJ.
      An update on the human and animal enteric pathogen Clostridium perfringens.
      ;
      • Flores-Díaz M
      • Alape-Girón A.
      Role of Clostridium perfringens phospholipase C in the pathogenesis of gas gangrene.
      ). However, after extensive literature review, only toxinotype A has been found in human cases with haemolysis to date. The fact that alpha toxin alone has been found is likely to be the result of testing just a small number of (typing) toxins in a small number of strains. Considering the known wide array of toxins and virulence factors which C. perfringens can produce, it would be shortsighted to conclude that alpha toxins alone play a virulent role. Thus, a complete overview of the possible pathogenic role of other toxinotypes and their toxins is lacking. Toxinotyping of future cases could reveal whether human haemolytic C. perfringens septicemia is indeed caused solely by type A strains.

      Conclusion

      Current treatment strategies are ineffective or initiated too late for most patients with haemolytic C. perfringens septicemia. As the alpha-toxin storm is the biggest driving force behind deterioration, the authors advocate the need to investigate supplemental treatment approaches in a toxin-specific way [e.g. antiserum (
      • Goossens E
      • Verherstraeten S
      • Valgaeren BR
      • Pardon B
      • Timbermont L
      • Schauvliege S
      • et al.
      The C-terminal domain of Clostridium perfringens alpha toxin as a vaccine candidate against bovine necrohemorrhagic enteritis.
      ) or nanobodies that target haemolytic activity]. Improved attention to determination and reporting of C. perfringens toxins and toxinotypes may provide more insight into the role of these toxins and virulence factors in pathophysiology, and could subsequently reveal new targets for intervention.

      Conflict of interest statement

      None declared.

      Funding

      None.

      Ethical approval

      Not required.

      Author contributions

      Study design: NW, JP and JL.
      Data collection: NW, JP, FI, EG, MB, CR, RS, IP and JL.
      Data analysis and writing: NW, JP, FI, EG and JL.
      All authors reviewed the manuscript and approved the final manuscript.

      References

        • Flores-Díaz M
        • Alape-Girón A.
        Role of Clostridium perfringens phospholipase C in the pathogenesis of gas gangrene.
        Toxicon. 2003; 42: 979-986
        • Goossens E
        • Verherstraeten S
        • Valgaeren BR
        • Pardon B
        • Timbermont L
        • Schauvliege S
        • et al.
        The C-terminal domain of Clostridium perfringens alpha toxin as a vaccine candidate against bovine necrohemorrhagic enteritis.
        Vet Res. 2016; 47: 52
        • Kiu R
        • Hall LJ.
        An update on the human and animal enteric pathogen Clostridium perfringens.
        Emerg Microbes Infect. 2018; 7: 141
        • McArthur HL
        • Dalal BI
        • Kollmannsberger C.
        Intravascular hemolysis as a complication of Clostridium perfringens sepsis.
        J Clin Oncol. 2006; 24: 2387-2388
        • Rood JI
        • Adams V
        • Lacey J
        • Lyras D
        • McClane BA
        • Melville SB
        • et al.
        Expansion of the Clostridium perfringens toxin-based typing scheme.
        Anaerobe. 2018; 53: 5-10
        • Simon TG
        • Bradley J
        • Jones A
        • Carino G.
        Massive intravascular hemolysis from Clostridium perfringens septicemia: a review.
        J Intensive Care Med. 2014; 29: 327-333
        • Stevens DL
        • Maier KA
        • Mitten JE.
        Effect of antibiotics on toxin production and viability of Clostridium perfringens.
        Antimicrob Agents Chemother. 1987; 31: 213-218
        • Stevens DL
        • Bryant AE.
        Role of clostridial toxins in the pathogenesis of gas gangrene.
        Clin Infect Dis. 2002; 35: S93-100
        • Stevens DL
        • Aldape MJ
        • Bryant AE.
        Life-threatening clostridial infections.
        Anaerobe. 2012; 18: 254-259
        • Suzaki A
        • Ohtani K
        • Komine-Aizawa S
        • Matsumoto A
        • Kamiya S
        • Hayakawa S.
        Pathogenic characterization of Clostridium perfringens strains isolated from patients with massive intravascular hemolysis.
        Front Microbiol. 2021; 12713509
        • van Bunderen CC
        • Bomers MK
        • Wesdorp E
        • Peerbooms P
        • Veenstra J.
        Clostridium perfringens septicaemia with massive intravascular haemolysis: a case report and review of the literature.
        Neth J Med. 2010; 68: 343-346
        • Watt J
        • Amini A
        • Mosier J
        • Gustafson M
        • Wynne JL
        • Friese R
        • et al.
        Treatment of severe hemolytic anemia caused by Clostridium perfringens sepsis in a liver transplant recipient.
        Surg Infect (Larchmt). 2012; 13: 60-62