Invasive Streptococcus pneumoniae infection causing hemolytic uremic syndrome in children : Two recent cases

1Alberta Children’s Hospital, Calgary Alberta; 2University of Calgary, Calgary, Alberta; 3National Centre for Streptococcus, Edmonton, Alberta Correspondence and reprints: Dr James D Kellner, Alberta Children’s Hospital, 1820 Richmond Road SW, Calgary, Alberta T2T 5C7. Telephone 403-943-7687, fax 403-943-7665, e-mail Jim.Kellner@CalgaryHealthRegion.ca Received for publication May 1, 2003. Accepted September 12, 2003 OG Vanderkooi, JD Kellner, AW Wade, et al. Invasive Streptococcus pneumoniae infection causing hemolytic uremic syndrome in children: Two recent cases. Can J Infect Dis 2003;14(6):339-343.


Invasive Streptococcus pneumoniae infection causing hemolytic uremic syndrome in children:
Two recent cases uncommon cause of hemolytic uremic syndrome (HUS) (1,2).The pathogenesis of S pneumoniae-associated HUS is different from Shiga toxin-associated HUS.The primary factor appears to be the production of neuraminidase by S pneumoniae (1).Patients treated with washed blood products tend to have a better outcome, likely related to decreased binding of naturally occurring antibodies to activated T antigen (3)(4)(5)(6)(7).The lack of a consistent case definition has resulted in debate about the distinction between cases of S pneumoniae-associated HUS and disseminated intravascular coagulation associated with invasive S pneumoniae infections, especially when diagnostic renal biopsy is not performed (8).
HUS is the most common cause of acute renal failure in children (3).Most cases are caused by Shiga-like toxins (9,10), and the etiology for Shiga-like toxin-associated HUS is usually enterohemorrhagic Escherichia coli 0157:H7 (3,9,11).However, many other E coli serotypes, as well as Shigella dysenteriae, Aeromonas species and the human immunodeficiency virus can cause HUS (9).In addition, other neuraminidase-producing organisms, such as influenza A virus, have been reported to cause HUS (12,13).Hereditary, drug-induced, disease-associated and idiopathic HUS have been reported (9,11).The disease is heralded by the sudden onset of microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure, often preceded by an acute gastroenteritis with bloody diarrhea (9,10).
We present two patients with S pneumoniae-associated HUS who presented to the Alberta Children's Hospital, Calgary, Alberta within one month of each other in 2001, and who were not epidemiologically linked.We also review the published literature concerning HUS associated with S pneumoniae.
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METHODS
A chart review was performed for each case.The organisms were identified from the clinical specimens using standard techniques, and antibiotic susceptibility testing was performed.Serotyping of the S pneumoniae isolates was performed by the Quellung method at the National Streptococcus Centre in Edmonton, Alberta.
Pulsed-field gel electrophoresis (PFGE) analysis was performed on both isolates.The Sma I restriction endonuclease was used in 1% agarose gel using 0.5 × tris-borate-ethylene diamine tetraacetic acid buffer at 10°C.The Chef Mapper (BioRad, USA) was used, and switch and run times were 0.20 to 35 s over 21 h, with a voltage gradient of 6.0 V/cm.
S pneumoniae exotoxin was determined by both toxicity assay and polymerase chain reaction (PCR) to identify the genes responsible for exotoxin production.The toxicity assay was performed using Vero cells.Controls used were S pneumoniae ATCC 27336, S pneumoniae ATCC 49619 and E coli pJB 128 (harbouring the VT1 operon on a multicopy plasmid, VT1 positive) (14).After overnight growth of the organism in brain heart infusion broth, 1.5 mL was removed and filtered using a Millex-GV syringe filter (Millipore, USA).This was then used for the exotoxin production assay.The plate was incubated in a humid chamber at 37°C and observed every 24 h for cytopathic effect.
PCR to identify Verotoxin genes was also performed.Control strains included S pneumoniae ATCC 27336 (negative control), an E coli 0157:H7 strain known to be VT1 positive (laboratory clinical isolate) and an E coli 0157:H -strain known to be VT2 positive (laboratory clinical isolate) (positive controls).A suspension containing one of the above organisms was pelleted, resuspended in 200 µL 10% Triton X-100 (Sigma, USA) and boiled for 20 min.The specimens and controls were then centrifuged for 20 min at 13,200 rpm, and the supernatant was used for the PCR.The following PCR conditions were used: hold for 15 min at 95°C, 25 cycles (denature for 40 s at 94°C, anneal for 40 s at 55°C, and elongate for 40 s at 72°C), hold for 6 min at 72°C and finally hold forever at 4°C.

CASE PRESENTATIONS
Case report 1 A two-year old, previously well, white boy presented with a seven-day history of upper respiratory symptoms with gradual worsening.He presented with fever, cough, vomiting, lethargy and decreasing urine output.There was no history of diarrhea, urinary tract infections, easy bruising or bleeding, travel, poorly cooked meats or unpasteurized diary products.He attended daycare and had a 10-month-old sibling with an upper respiratory tract infection.Family history was noncontributory.
On admission, he was afebrile (however, fever had been noted by family), with a pulse of 150 beats/min and a blood pressure of 120/80 mmHg.His respiratory rate was 50 breaths/min with significantly increased effort.He was moderately dehydrated.His oxygen saturation remained normal in room air.Chest examination revealed dullness to percussion and decreased breath sounds on the right.Jaundice and pallor were also present.A chest x-ray showed right middle and lower lobe consolidation and a right-sided effusion.
His hemoglobin level was 95 g/L, which decreased to 67 g/L after rehydration.Platelets were 15×10 9 /L, with a normal white blood cell count of 4.8×10 9 /L.The erythrocyte sedimentation rate was 148 mm/h.His creatinine level was 77 µmol/L and urea level was 21 mmol/L.The bilirubin and lactate dehydrogenase concentrations were 145 µmol/L and 2805 U/L, respectively.Serum cold agglutinins were strongly positive.There was gross hematuria, and the urinalysis demonstrated proteinuria.A smear of the peripheral blood demonstrated fragmented red blood cells and schistocytes consistent with microangiopathic hemolytic anemia.The partial thromboplastin time was 74 s (normal 25 to 35 s), international normalized ratio (INR) was 1.1, D-dimers were 0.2 to 0.4 mg/L (normal less than 0.2 mg/L) and fibrinogen was 9.3 g/L (normal 2 to 4 g/L).Cefotaxime and vancomycin were started for empiric therapy.Blood cultures and subsequent pleural fluid obtained by thoracocentesis grew S pneumoniae, serotype 3 -susceptible to penicillin, cefotaxime and vancomycin.Pleural fluid chemistry revealed a concentration of glucose less than 0.5 mmol/L, a concentration of lactate dehydrogenase greater than 4500 U/L, a pH of 7.73 and a protein level of 35 g/L.Urine culture was negative.Acute and convalescent serology for Mycoplasma pneumoniae was also negative.Stool cultures were negative for E coli 0157 and Shigella species, but serology was positive (titre greater than 1:128) for antibody to Shiga toxin 2 by neutralization assay by the National Laboratory for Public Health, Winnipeg, Manitoba.This may have indicated past exposure.After antibiotic susceptibilities were known, the antibiotic treatment was changed to intravenous penicillin G.
His course in hospital included six washed, packed red blood cell transfusions, five washed platelet transfusions, nine days of hemodialysis and an additional nine days of peritoneal dialysis.His course was complicated by the empyema, treated conservatively without chest drain, pancreatitis (lipase level 3041 U/L) and hepatitis (aspartate aminotransferase level 800 U/L and alanine aminotransferase level 269 U/L).At 11 months following admission, his renal function was normal, with a creatinine concentration of 42 µmol/L and a calculated creatinine clearance of 95 mL/min/1.73m 2 .

Case report 2
A four-year-old, previously well, white boy presented with intractable epistaxis and a seven-day history of upper respiratory symptoms, including sore throat.During the five days preceding admission, symptoms of abdominal pain, vomiting, increasing fevers with progressive tachypnea and dyspnea were present.He had no history of diarrhea, fast food or poorly cooked meat products.There was no history of travel.His mother and sister also had upper respiratory infections.His immunizations were incomplete.Family history was significant for urinary tract infections but not renal disease.
On admission, he was afebrile, with a pulse of 159 beats/min and a blood pressure of 105/67 mmHg.His respiratory rate was 55 breaths/min with obvious increased effort.He was moderately dehydrated.His oxygen saturation was normal in room air.Chest examination revealed dullness to percussion with decreased breath sounds to the left hemithorax.Jaundice and pallor were also present.A chest x-ray confirmed left lower lobe consolidation with effusion.
On admission, his hemoglobin level was 69 g/L.His platelet count was 4×10 9 /L and his white blood cell count was 13.7×10 9 /L, with 52% neutrophils and 29% band forms.The erythrocyte sedimentation rate was 148 mm/h.His creatinine level was 110 µmol/L and his urea level was 20 mmol/L.The total bilirubin and lactate dehydrogenase concentrations were 75 µmol/L (20 µmol/L conjugated) and greater than 4500 U/L, respectively.The aspartate aminotransferase level was 400 U/L, and the alanine aminotransferase level was normal.The urine demonstrated gross hematuria and proteinuria.A smear of the peripheral blood demonstrated fragmented red blood cells and schistocytes consistent with microangiopathic hemolytic anemia.The patient's partial thromboplastin time was 47 s, INR was 1.4, D-dimers were 0.8 to 1.6 mg/L and fibrinogen was 5.1 g/L.Cefotaxime and vancomycin were empirically started.
Blood cultures and subsequent pleural fluid cultures grew S pneumoniae, serotype 14 -susceptible to penicillin, cefotaxime and vancomycin.A urine culture was negative.A throat swab for group A beta-hemolytic streptococcus was negative and the antistreptolysin O titre was negative.Stool cultures were not sent, but serology for Shiga toxins 1 and 2 were negative by neutralization assay.After antibiotic susceptibilities were known, antibiotic treatment was changed to intravenous penicillin G.
His course in hospital included five washed, packed red blood cell transfusions, four washed platelet transfusions and 10 days of hemodialysis.He required nasal cauterization and packing; the clinical course was further complicated by empyema, hypertension, pericarditis and an incidental finding of Wolff-Parkinson-White syndrome diagnosed after an episode of tachycardia in the perioperative period.
At nine months postadmission, his renal function was normal, with a creatinine of 33 µmol/L and a calculated creatinine clearance of 132 mL/min/1.73m 2 .
The S pneumoniae isolates from both cases were unique on PFGE analysis.Neither organism showed evidence of Shiga toxin production nor evidence of Shiga toxin-encoding genes.

DISCUSSION
The presumed mechanism of pathogenesis for HUS associated with S pneumoniae differs from Shiga toxin-related disease (1,33).The Thomsen Friedenreich antigen (T antigen) is a normal structure on the surface of erythrocytes and renal capillary endothelial cells that is obscured by neuramic acid (1,19,34).The neuraminidase produced by S pneumoniae (35,36) circulates in serum (6,19,37) and removes neuraminic acid from glycoproteins, revealing the T antigen on cell surfaces (1,6,35).This leads to binding with T antigen-specific immunoglobulin (Ig) M, which is present in all human serum by the age of six months (1,19,34,35), resulting in hemolysis (1,35), agglutination (27) and microangiopathy in the kidneys (1,35) with secondary thrombocytopenia.In such cases, circulating serum anti-T antigen IgM levels may be reduced to zero as a result of consumption (19).
This activated T antigen is also present on hepatocytes and may contribute to the hepatic dysfunction noted in some case series (10,16); as well, T antigen found in the choroid plexus in the brain may contribute to the pathogenesis of meningitis (38,39).Patients with S pneumoniae-associated HUS have demonstrable neuraminidase activity, while those with invasive S pneumoniae disease without HUS do not (6).
Early recognition of S pneumoniae-associated HUS is important -plasma or plasma-containing blood products may worsen the clinical course, because most healthy individuals contain anti-T IgM in their serum (6,19,40).Fluorescein-labelled peanut agglutinin (Arachis hypogaea) confirms the presence of T antigen on tested cells or tissues (35,41).Although this assay is a straightforward test, it is not routinely performed in many laboratories (Dr D Easton, Calgary Laboratory Services, Calgary, Alberta August 2002, personal communication).The direct Coombs test is another test that detects (auto-) antibody binding to red blood cells, and a previous, brief review of cases of HUS associated with S pneumoniae noted that when performed in these cases, the direct Coombs test is usually positive (42).It has been suggested that this test, which is more readily available, could be useful to detect HUS early in cases of invasive S pneumoniae disease (42).However, there are no data on the specificity of the direct Coombs test in these cases.
It is uncertain how often HUS occurs in association with S pneumoniae infections.A review of cases from Atlanta found that HUS occurred after 0.6% of invasive S pneumoniae infections (3).In contrast, HUS develops after 5% to 14% of E coli 0151:H7 infections (43,44).
There was significant mortality in the published cases of S pneumoniae-associated HUS, with 15 deaths among 72 cases (21% of the total of the 70 cases previously reported in the literature and the two cases reported in the present study) (1,3,6,7,11,19,20,32).These deaths were reported in publications from 1977 to 2001, and the cause of death was not always described, but when described, it was related to acute or chronic renal disease, or to severe acute S pneumoniae infection.Thus, it cannot readily be determined whether there is a trend in the incidence of case fatalities related to changes in the care provided to these children.The use of washed blood products has been shown to improve outcome (3-7); earlier treatments included exchange transfusion (19,35).
Although our two patients recovered normal renal function, among reported survivors, there are a considerable number of children with ongoing renal problems; many went on to end-stage renal failure (2,4,5,10,11,15,16,18,31).
Most authors did not comment on the coagulation profiles in their cases.However, some authors did note similar, mild abnormalities in the coagulation profiles seen in their cases, which were similar to the present cases (3,5,16,25,30,31).It is unclear whether these mild abnormalities are part of a systemic inflammatory response to the S pneumoniae infection or are a result of HUS.
Both of our patients had mild liver function abnormalities.This has been described in several patients as part of their clinical presentation (5,10).
There is variability in the laboratory criteria for reported cases.Of the 70 reported cases, only 16 had clear demonstration of T antigen with agglutination on red blood cells (4)(5)(6)(7)10,16,19,27), seven had data about T antigen agglutination on kidney tissue (1,6,15,26,27) and six had data about thrombotic microangiopathy on kidney tissues (11).The presence of T antigen on cell surfaces and its clinical implications warrant further investigation; however, it is not clear what kind of T antigen test may be appropriate as a routine test.
There is no consistent case definition for S pneumoniaeassociated HUS.The Canadian Paediatric Society has a surveillance definition, and the Centers for Disease Control and Prevention currently have a case definition for postdiarrheal HUS (47), but not for S pneumoniae-associated HUS alone.The Canadian Paediatric Surveillance Program case definition is outlined in Table 1.From April 2000 to March 2002, three possible cases and one definite case were reported in Canada (48) using the above definitions, and debate continues as to which cases truly qualify as S pneumoniae-associated HUS (3,8).
We suggest a modification to the Canadian Pediatric Society case definition so that 'possible' cases are considered to be 'probable' cases.Renal biopsy is not usually necessary to make a diagnosis of S pneumoniae-associated HUS if all other criteria have been fulfilled and if clinicians are confident that the laboratory changes are not caused by sepsis.The diagnosis can be based on findings consistent with microangiopathic hemolytic anemia on a peripheral blood smear, in addition to the biochemical renal and hematological abnormalities noted in Table 1.In circumstances in which the diagnosis or etiology is unclear, a biopsy could be considered.
If a widely accepted case definition can be determined for S pneumoniae-associated HUS, further studies about the clinical course, as well as the therapeutic and prognostic value of measuring neuraminidase activity, T antigen and T antigen antibody should be facilitated.

TABLE 1 Canadian
Paediatric Society Streptococcus pneumoniaeassociated hemolytic uremic syndrome case definitions Children under the age of 16 years demonstrating the following (need not be present simultaneously): 1) Evidence of invasive S pneumoniae infection (blood or another normally sterile biological fluid: cerebrospinal, pericardial, articular, peritoneal, pleural), excluding middle ear, sinus and tracheal aspirates 2) Both renal and hematological organ failures defined as: a) Acute renal impairment with serum creatinine: >50 µmol/L if younger than five years old >60 µmol/L if five to nine years old >90 µmol/L if 10 to 13 years old >110 µmol/L if older than 13 years old b) Microangiopathic hemolytic anemia (hemoglobin <100g/L with fragmented red cells) c) Thrombocytopenia (<150,000×10 9 /L) in the absence of septicemia, malignant hypertension, chronic uremia, collagen or vascular disorders 3) No chronic conditions that are causative for the renal and hematological abnormalities seen Other organ failures may occur Definite case Evidence of thrombotic microangiopathy on renal biopsy or autopsy Possible case Distinction between pneumococcal sepsis with secondary organ failures and S pneumoniae-associated hemolytic uremic syndrome will be determined through a Delphi process Data taken from reference 49