Hemolytic Uremic Syndrome following O111 Shiga toxin producing

Hemolytic Uremic Syndrome following O111 Shiga toxin producing E. coli revealed 1

through molecular diagnostics 2

3

Darwin J. Operario1, Shannon Moonah1, Eric Houpt1* 4

5

1-University of Virginia Health System, Division of Infectious Diseases and International 6

Health, Charlottesville, Virginia, USA 7

8

*Corresponding Author: Eric Houpt <[email protected]> 9

10

JCM Accepts, published online ahead of print on 26 December 2013J. Clin. Microbiol. doi:10.1128/JCM.02855-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 11

We report a case of hemolytic uremic syndrome in a 69-year old woman due to Shiga toxin-12

producing Escherichia coli, possibly serotype O111, to illustrate the potentially deleterious 13

implications of a Campylobacter EIA result and the increasing importance of molecular testing 14

when conventional methods are limited. 15

16

17

CASE REPORT 18

A 69-year old female presented to our hospital in July 2013 with a two day history of abdominal 19

cramping and hematochezia after a camping trip. During the trip the patient reported consuming 20

fresh produce and store-bought bottled water, but no meat, poultry, or eggs. Reportedly none of 21

the other 40 people on the camping trip were ill. Examination revealed an afebrile and 22

tachycardic (pulse rate 118 beats/minute) woman with a diffusely tender abdomen on deep 23

palpation. Initial stool studies were positive for lactoferrin and Campylobacter by EIA (Meridian 24

Biosciences Inc., Cincinnati, OH), negative for C. difficile PCR and Giardia antigen, while Shiga 25

toxin testing (EIA, Meridian Biosciences) was pending. Azithromycin was empirically initiated 26

for presumed infectious colitis due to Campylobacter spp. The next day the Shiga toxin test 27

returned positive and the patient developed worsening bloody diarrhea and leukocytosis (white 28

cell count 24 x 103/ mm3) and lactic acidosis. Abdominal-pelvic CT scan revealed severe bowel 29

wall thickening throughout the entire length of the colon. She was transferred to the intensive 30

care unit for further monitoring and management. Ciprofloxacin and metronidazole were added 31

for severe colitis. Thrombocytopenia developed (platelet count 68 x 103/mm3) after which all 32

antimicrobial therapy was discontinued as hemolytic uremic syndrome (HUS) was suspected. 33


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The patient went on to develop hemolytic anemia with schistocytosis, acute renal failure, and 34

altered mental status changes. The patient was supported with plasmapharesis and hemodialysis 35

therapy. An additional stool sample was obtained 2 days after admission which was 36

Campylobacter EIA negative and also Shiga toxin negative in broth culture. The initial broth was 37

sent to the Virginia Division of Consolidated Laboratory Services (DCLS) and resulted in no 38

culturable bacteria but was PCR positive for stx1 and negative for stx2 and uidA genes (Table 2). 39

We developed and utilized two multiplex real-time PCR reactions targeting STEC-associated 40

genes: one panel for eae, stx1, stx2, and rfbEO157, and a second panel for wzx or CRISPR genes of 41

relevant non-O157 serotypes. These assays confirmed the presence of stx1, and detected both the 42

wzx gene of O111 and eae (Table 2). All multiplex real-time results were confirmed in 43

singleplex. The wzx amplicon was sequenced and revealed a perfect match with 50 available 44

O111-specific wzx sequences within NCBI (representing both STEC and non-STEC sources). 45

Molecular testing for Campylobacter spp. (16S) and C. jejuni/coli (cadF, (8)) were negative. 46

While we acknowledge that the detected genes could have originated from different organisms 47

and that the wzx sequences detected here are not STEC-specific, based on the detected molecular 48

profile (stx1, eae, wzxO111 positive) we believe the most likely scenario is that the patient had E. 49

coli O111 STEC-HUS. The patient recovered completely and was discharged home after a 50

lengthy 23 day admission. 51

52

53

Shiga toxin-producing E. coli (STEC; also known as verotoxin-producing E. coli, VTEC) are a 54

notable group of foodborne pathogens due to their capability for producing foodborne outbreaks 55

of bloody diarrhea as well as the systemic complication of HUS. STEC caused a total of 308 56


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laboratory-confirmed outbreaks in the United States between 1998-2008 (14). The most 57

commonly recognized of these STEC serotypes is E. coli O157:H7, with greater than 63,000 58

estimated episodes each year in the United States (19). However, non-O157 strains have been 59

increasingly implicated. FoodNet surveillance data from 2007 indicated an incidence rate of 60

1.20 per 100,000 for O157 STEC with a rate of 0.57 for non-O157 STEC (2). The six O26, O45, 61

O103, O111, O121, and O145 serogroups accounted for 71% of all non-O157 STEC isolates 62

submitted to the CDC between 1983 and 2002 (6). In June, 2012, the United States Department 63

of Agriculture (USDA) declared these six serogroups as adulterants in ground beef (21). 64

Serotype O111 in particular has been implicated in several outbreaks both within and outside the 65

U.S. (1, 9, 18, 20). Of note the STEC causing an outbreak of bloody diarrhea and HUS in 66

Germany during 2011 was serotype O104:H4 (4, 5). 67

68

Detection and isolation of non-O157 STEC strains from clinical specimens can be challenging 69

because non-O157 STEC strains often lack phenotypic characteristics (such as sorbitol 70

fermentation) that would distinguish them from non-pathogenic E. coli strains (21). STEC 71

strains are characterized by the presence of Shiga toxin produced from genes stx1 and/or stx2. 72

EIA or PCR methods to detect the toxin itself or the cognate genes are available (7). However 73

this provides little epidemiologic information, nor does detection of other virulence factors such 74

as the intimin gene, eae, or the hemolysin gene, ehxA (3, 10, 11). The current method employed 75

by the USDA for food safety uses a multiplex PCR for stx1, stx2, and eae before using other 76

genetic markers, such as variations in the wzx gene, to screen for O26, O45, O103, O111, O121, 77

or O145 (12). 78

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Based on this, we developed a pair of assay panels to screen putative E. coli outbreak samples. 80

The first panel detects stx1, stx2, eae, and rfbEO157, along with a Phocine Herpes Virus (PhHV) 81

extraction/amplification control. The second panel detects serotype-specific wzx (O26, O103, 82

O111, O145) or CRISPR (O45, O103, O121) gene segments. We evaluated the assay panels on a 83

set of non-O157 E. coli isolates from the Virginia Division of Consolidated Laboratory Services 84

as well as the O157:H7 reference strain 43895 (ATCC, Manassas, VA). 85

86

In our case of probable STEC O111 infection with HUS, initial antibiotic therapy was based on a 87

positive Campylobacter stool antigen test. Campylobacter was not confirmed by culture or PCR, 88

suggesting that the initial Campylobacter EIA was likely a false-positive result. Clinicians and 89

clinical labs should thus be cautious when interpreting Campylobacter EIA results in a possible 90

STEC scenario (13). This case report shows the utility of molecular testing to diagnose infections 91

where conventional methods may fail and such results can be clinically valuable or used by 92

public health laboratories for refined epidemiology of STEC. 93

94

ACKNOWLEDGEMENTS 95

We would like to thank Denise Toney and staff members at the Virginia Division of 96

Consolidated Laboratory Services for their work in the clinical sample evaluation and for 97

materials support during the development of the multiplex assays. 98


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TABLE 1 E. coli Molecular Assays Utilized at UVA

Table 1A, Panel 1

Gene Oligonucleotide sequences* (5’3’), dyes, quenchers,

final concentration in reaction

LOD¶ in

Stool/ GN

Broth

Reference

stx1

F: ACTTCTCGACTGCAAAGACGTATG, 0.2µM

R: ACAAATTATCCCCTGAGCCACTATC, 0.2µM

P: FAM-CTCTGCAATAGGTACTCCA-MGBBHQ1, 0.2µM

104/103 (15)

stx2

F: CCACATCGGTGTCTGTTATTAACC, 0.2µM

R: GGTCAAAACGCGCCTGATAG, 0.2µM

P: VIC-TTGCTGTGGATATACGAGG-MGBBHQ2, 0.2µM

104/103 (15)

eae

F: CATTGATCAGGATTTTTCTGGTGATA, 0.15µM

R: CTCATGCGGAAATAGCCGTTA, 0.15µM

P: Quasar705-ATACTGGCGAGACTATTTCAA-BHQ2,

0.2µM

104/103 (17)

rfbEO157

F: TTTCACACTTATTGGATGGTCTCAA, 0.4µM

R: CGATGAGTTTATCTGCAAGGTGAT, 0.4µM

P: Texas Red-

AGGACCGCAGAGGAAAGAGAGGAATTAAGG-

BHQ2, 0.1µM

104/103 (16)

PhHV

F: GGGCGAATCACAGATTGAATC, 0.6µM

R: GCGGTTCCAAACGTACCAA, 0.6µM

P: Quasar 670-TTTTTATGTGTCCGCCACCATCTGGATC-

BHQ2, 0.2µM

(17)


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Reactions were performed on a Bio-Rad CFX96 qPCR system (Bio-Rad, Hercules, CA) in a 25µL reaction volume

using Bio-Rad iQ Multiplex Powermix and 5µL DNA extract; Cycling conditions were as follows: 3 minutes 95°C

followed by 40 cycles of 95°C for 10 seconds and 60°C for 1 minute.

*: Unmodified oligonucleotides and non-MGB dual-labeled probes were obtained from Integrated DNA

Technologies, Inc. (IDT, Coralville, IA); MGB dual-labeled probes were obtained from Life Technologies/Applied

Biosystems (Applied Biosystems, Foster City, CA)

¶: Limit of Detection


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Table 1B, Panel 2

STEC target/

Gene Oligonucleotide* sequences (5’3’)

Amplicon Tm on

CFX96, °C Reference

O26, wzx F: GTATCGCTGAAATTAGAAGCGC

R: CTTATGGACAATCCAACCGA 77.0

(12),

modified

O45/O103,

CRISPR

F: GAGTCTATCAGCGACACTACC

R: AACCGCAGCTCGCAGCGC 88.0 (10)

O103, wzx F: TTGGAGCGTTAACTGGACCT

R: ATATTCGCTATATCTTCTTGCGGC 80.5 (12)

O111, wzx F: TGTTCCAGGTGGTAGGATTCG

R: ACTTCCTGAAATACCATACTCT 74.5

(12),

modified

O121,

CRISPR

F: CGGGGAACACTACAGGAAAGAA

R: GGCGGAATACAGGACGGGTGG 86.5 (10)

O145, wzx F: AAACTGGGATTGGACGTGG

R: GCGAATCTATCAAACGTGAA 80.5

(12),

modified

Reactions were performed on a Bio-Rad CFX96 qPCR system (Bio-Rad) in a 25µL reaction volume using

AccuStart II PCR ToughMix (Quanta Biosciences, Gaithersburg, MD) supplemented with EVA Green (Biotium,

Hayward, CA) at a final concentration of 0.5x and 5µL DNA extract; Cycling conditions were as follows: 3 minutes

95°C followed by 40 cycles of 95°C for 10 seconds, 61°C for 15 seconds, 72°C for 15 seconds; followed by a

melting curve of 65°C to 95°C in 0.5°C increments and dwelling at each temperature for 5 seconds. The identity of a

particular STEC was determined using the Tm of the amplicon through melt analysis.

*: All oligonucleotides in this reaction were used at 0.4µM final concentration and were obtained from Integrated

DNA Technologies, Inc. (IDT)


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TABLE 2 Molecular results in this case

UVA testing DCLS testing

Gene Result, Ct Result

stx1 +, 21.1 +

stx2 -* -

eae +, 21.1 nd

rfbEO157 -* nd

uidAO157 nd -

O26, wzx - nd

O45/O103,

CRISPR - nd

O103, wzx - nd

O111, wzx +, 23.7, Tm at 74.5°C nd

O121,

CRISPR - nd

O145, wzx - nd

*negative was defined as no amplification or Ct at or beyond limit of detection (stx2 had Ct of 37 and rfbE had a Ct

of 36, both of these results were at LOD and we interpreted as negative; no other amplification observed)

nd=not done.


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Hemolytic Uremic Syndrome following O111 Shiga toxin producing