Hemolytic Uremic Syndrome following O111 Shiga toxin producing ...
Transcript of Hemolytic Uremic Syndrome following O111 Shiga toxin producing ...
Hemolytic Uremic Syndrome following O111 Shiga toxin producing E. coli revealed 1
through molecular diagnostics 2
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Darwin J. Operario1, Shannon Moonah1, Eric Houpt1* 4
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1-University of Virginia Health System, Division of Infectious Diseases and International 6
Health, Charlottesville, Virginia, USA 7
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*Corresponding Author: Eric Houpt <[email protected]> 9
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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
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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
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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
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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
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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
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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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1. 2012. Outbreak of Shiga toxin-producing Escherichia coli O111 infections associated 99
with a correctional facility dairy - Colorado, 2010. MMWR. Morbidity and Mortality 100
Weekly Report 61:149-152. 101
2. 2008. Preliminary FoodNet data on the incidence of infection with pathogens transmitted 102
commonly through food--10 states, 2007. MMWR. Morbidity and Mortality Weekly 103
Report 57:366-370. 104
3. Bai, J., Z. D. Paddock, X. Shi, S. Li, B. An, and T. G. Nagaraja. 2012. Applicability 105
of a multiplex PCR to detect the seven major Shiga toxin-producing Escherichia coli 106
based on genes that code for serogroup-specific O-antigens and major virulence factors in 107
cattle feces. Foodborne Pathogens and Disease 9:541-548. 108
4. Bielaszewska, M., A. Mellmann, W. Zhang, R. Kock, A. Fruth, A. Bauwens, G. 109
Peters, and H. Karch. 2011. Characterisation of the Escherichia coli strain associated 110
with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological 111
study. The Lancet Infectious Diseases 11:671-676. 112
5. Bloch, S. K., A. Felczykowska, and B. Nejman-Falenczyk. 2012. Escherichia coli 113
O104:H4 outbreak--have we learnt a lesson from it? Acta Biochimica Polonica 59:483-114
488. 115
6. Brooks, J. T., E. G. Sowers, J. G. Wells, K. D. Greene, P. M. Griffin, R. M. 116
Hoekstra, and N. A. Strockbine. 2005. Non-O157 Shiga toxin-producing Escherichia 117
coli infections in the United States, 1983-2002. The Journal of Infectious Diseases 118
192:1422-1429. 119
7. Clogher, P., S. Hurd, D. Hoefer, J. L. Hadler, L. Pasutti, S. Cosgrove, S. Segler, M. 120
Tobin-D'Angelo, C. Nicholson, H. Booth, K. Garman, R. K. Mody, and L. H. Gould. 121
on April 4, 2018 by guest
http://jcm.asm
.org/D
ownloaded from
2012. Assessment of physician knowledge and practices concerning Shiga toxin-122
producing Escherichia coli infection and enteric illness, 2009, Foodborne Diseases 123
Active Surveillance Network (FoodNet). Clin Infect Dis 54 Suppl 5:S446-452. 124
8. Cunningham, S. A., L. M. Sloan, L. M. Nyre, E. A. Vetter, J. Mandrekar, and R. 125
Patel. 2010. Three-hour molecular detection of Campylobacter, Salmonella, Yersinia, 126
and Shigella species in feces with accuracy as high as that of culture. Journal of Clinical 127
Microbiology 48:2929-2933. 128
9. Dallman, T., G. P. Smith, B. O'Brien, M. A. Chattaway, D. Finlay, K. A. Grant, and 129
C. Jenkins. 2012. Characterization of a verocytotoxin-producing enteroaggregative 130
Escherichia coli serogroup O111:H21 strain associated with a household outbreak in 131
Northern Ireland. Journal of Clinical Microbiology 50:4116-4119. 132
10. Delannoy, S., L. Beutin, and P. Fach. 2012. Use of clustered regularly interspaced short 133
palindromic repeat sequence polymorphisms for specific detection of enterohemorrhagic 134
Escherichia coli strains of serotypes O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, 135
O145:H28, and O157:H7 by real-time PCR. Journal of Clinical Microbiology 50:4035-136
4040. 137
11. Fratamico, P. M., and L. K. Bagi. 2012. Detection of Shiga toxin-producing 138
Escherichia coli in ground beef using the GeneDisc real-time PCR system. Frontiers in 139
Cellular and Infection Microbiology 2:152. 140
12. Fratamico, P. M., L. K. Bagi, W. C. Cray, Jr., N. Narang, X. Yan, M. Medina, and 141
Y. Liu. 2011. Detection by multiplex real-time polymerase chain reaction assays and 142
isolation of Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, 143
O121, and O145 in ground beef. Foodborne Pathogens and Disease 8:601-607. 144
on April 4, 2018 by guest
http://jcm.asm
.org/D
ownloaded from
13. Giltner, C. L., S. Saeki, A. M. Bobenchik, and R. M. Humphries. 2013. Rapid 145
detection of Campylobacter antigen by enzyme immunoassay leads to increased 146
positivity rates. Journal of Clinical Microbiology 51:618-620. 147
14. Gould, L. H., K. A. Walsh, A. R. Vieira, K. Herman, I. T. Williams, A. J. Hall, and 148
D. Cole. 2013. Surveillance for foodborne disease outbreaks - United States, 1998-2008. 149
MMWR Surveill Summ 62:1-34. 150
15. Hidaka, A., T. Hokyo, K. Arikawa, S. Fujihara, J. Ogasawara, A. Hase, Y. Hara-151
Kudo, and Y. Nishikawa. 2009. Multiplex real-time PCR for exhaustive detection of 152
diarrhoeagenic Escherichia coli. Journal of Applied Microbiology 106:410-420. 153
16. Jenkins, C., A. J. Lawson, T. Cheasty, and G. A. Willshaw. 2012. Assessment of a 154
real-time PCR for the detection and characterization of verocytotoxigenic Escherichia 155
coli. Journal of Medical Microbiology 61:1082-1085. 156
17. Liu, J., J. Gratz, C. Amour, G. Kibiki, S. Becker, L. Janaki, J. J. Verweij, M. 157
Taniuchi, S. U. Sobuz, R. Haque, D. M. Haverstick, and E. R. Houpt. 2013. A 158
laboratory-developed TaqMan Array Card for simultaneous detection of 19 159
enteropathogens. Journal of Clinical Microbiology 51:472-480. 160
18. Piercefield, E. W., K. K. Bradley, R. L. Coffman, and S. M. Mallonee. 2010. 161
Hemolytic Uremic Syndrome After an Escherichia coli O111 Outbreak. Archives of 162
Internal Medicine 170:1656-1663. 163
19. Scallan, E., R. M. Hoekstra, F. J. Angulo, R. V. Tauxe, M. A. Widdowson, S. L. Roy, 164
J. L. Jones, and P. M. Griffin. 2011. Foodborne illness acquired in the United States--165
major pathogens. Emerging Infectious Diseases 17:7-15. 166
on April 4, 2018 by guest
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20. Schaffzin, J. K., F. Coronado, N. B. Dumas, T. P. Root, T. A. Halse, D. J. 167
Schoonmaker-Bopp, M. M. Lurie, D. Nicholas, B. Gerzonich, G. S. Johnson, B. J. 168
Wallace, and K. A. Musser. 2012. Public health approach to detection of non-O157 169
Shiga toxin-producing Escherichia coli: summary of two outbreaks and laboratory 170
procedures. Epidemiology and Infection 140:283-289. 171
21. Wang, F., Q. Yang, J. A. Kase, J. Meng, L. M. Clotilde, A. Lin, and B. Ge. 2013. 172
Current Trends in Detecting Non-O157 Shiga Toxin-Producing Escherichia coli in Food. 173
Foodborne Pathogens and Disease 10:665-677. 174
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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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