Laboratory Practices for the Identification of Shiga Toxin–Producing Escherichia coli in the United States,FoodNet Sites,2007

Abstract

Clinical laboratory practices affect patient care and disease surveillance. It is recommended that laboratories routinely use both culture for Escherichia coli O157 and a method that detects Shiga toxins (Stx) to identify all Stx-producing E. coli (STEC) and that labs send broths or isolates to a public health laboratory. In 2007, we surveyed laboratories serving Foodborne Diseases Active Surveillance Network sites that performed on-site enteric disease diagnostic testing to determine their culture and nonculture-based testing practices for STEC identification. Our goals were to measure changes over time in laboratory practices and to compare reported practices with published recommendations. Overall, 89% of laboratories used only culture-based methods, 7% used only Stx enzyme immunoassay (EIA), and 4% used both Stx EIA and culture-based methods. Only 2% of laboratories reported simultaneous culture for O157 STEC and use of Stx EIA. The proportion that ever used Stx EIA increased from 6% in 2003 to 11% in 2007. The proportion that routinely tested all specimens with at least one method was 66% in 2003 versus 71% in 2007. Reference laboratories were less likely than others to test all specimens routinely by one or more of these methods (48% vs. 73%, p = 0.03). As of 2007, most laboratories complied with recommendations for O157 STEC testing by culture but not with recommendations for detection of non-O157 STEC. The proportion of laboratories that culture stools for O157 STEC has changed little since 2003, whereas testing for Stx has increased.

Introduction

S higa toxin–producing Escherichia coli (STEC) infection causes severe diarrhea, which is frequently bloody, and may result in life-threatening hemolytic uremic syndrome (Besser and Griffin, 1999). E. coli O157:H7 (O157) is the most commonly identified serotype of STEC in the United States (CDC, 2009). However, studies suggest that infection with STEC serogroups that express an O antigen other than O157 (non-O157 STEC) may be as prevalent (Andreoli et al., 2002; Klein et al., 2002) and has been associated with outbreaks (Brooks et al., 2005). In 2007, the Foodborne Diseases Active Surveillance Network (FoodNet) reported 272 cases of culture-confirmed non-O157 STEC or 0.57 infections per 100,000 persons compared with 1.2 per 100,000 for culture-confirmed O157 STEC (CDC, 2008).

Clinical laboratories can readily detect O157 STEC in stool specimens through the use of sorbitol-containing selective media, because most O157 STEC cannot ferment sorbitol within 24 hours. No selective or differential media are available to specifically detect non-O157 STEC. Most non-O157 STEC strains are indistinguishable from E. coli strains present in the normal flora by usual culture-based methods, as all ferment sorbitol.

Nonculture-based tests to detect Shiga toxins (Stx) in stool, typically enzyme immunoassays (EIA), were first licensed in 1995 and are being increasingly used by clinical laboratories. While a practical tool for STEC diagnosis, use of the Stx EIA without simultaneous or subsequent culture for O157 STEC delays or prevents further characterization of isolates and does not provide complete information for clinical decision making, as there is substantial variability in clinical outcome among the different STEC serogroups (Johnson et al., 2006; Persson et al., 2007).

Most clinical laboratories do not conduct Stx EIA testing; therefore, the burden of non-O157 STEC infections remains undefined (Cohen et al., 2008; Voetsch et al., 2004). Moreover, lack of uniformity exists among clinical laboratories regarding types of stool specimens cultured for O157 STEC or tested for Stx. For example, some laboratories will culture or test all stool specimens; others test only bloody stools; and others culture or test only on physician request (Voetsch et al., 2004). In 2006, CDC published recommendations for STEC testing to improve both routine surveillance and outbreak response (CDC, 2006), including the importance of testing all stool specimens.

In 2007, FoodNet surveyed all clinical laboratories that perform enteric disease diagnostic testing for residents of the FoodNet sites to determine their culture-based and nonculture-based laboratory testing practices for the identification of STEC. The goals of this study were to measure changes in laboratory practices of clinical laboratories serving the FoodNet catchment area and to compare reported practices with published diagnostic recommendations.

Materials and Methods

FoodNet is a collaborative sentinel site surveillance program conducted under the auspices of the Centers for Disease Control and Prevention's Emerging Infections Program (Angulo et al., 1998). FoodNet surveillance is conducted in 10 sites: the entire states of Connecticut, Georgia, Maryland, Minnesota, Oregon, Tennessee, and selected counties in California, Colorado, and New York According to the U.S. Census Bureau population estimates for 2007, the population of the FoodNet surveillance area was ∼45.5 million persons or 15% of the U.S. population. As part of surveillance activities, FoodNet personnel routinely contact all clinical laboratories serving the FoodNet sites for complete ascertainment of all cases. To better understand the practices of clinical laboratories, FoodNet also administers periodic surveys to laboratories serving the catchment area.

The survey was conducted during May–July 2007. FoodNet staff in each site identified all clinical laboratories performing enteric disease diagnostic testing for residents of their respective catchment areas. The survey was administered by an in-person visit, over the telephone, or via the internet. All clinical laboratories were first contacted by telephone, an e-mail, or a letter to introduce the survey. The lead microbiologist responsible for STEC testing was asked to participate in the survey. After the survey was completed, FoodNet staff coded each laboratory as being either a reference or nonreference type. Reference laboratories were defined as those not located in, operated by, or affiliated with a particular hospital or hospital system. State public health laboratories (SPHLs) were not surveyed.

The survey contained questions on the process of STEC testing, including under what circumstances culture versus nonculture-based methods are used, the specific nonculture-based tests used (e.g., EIA), and what follow-up procedures were used for positive results. Questions regarding follow-up procedures included topics such as subsequent testing of Stx EIA positive specimens, the frequency of submission to public health laboratories (e.g., batched), and how they are sent. Laboratories that used nonculture-based methods were asked the year they started using that method, and laboratories that used only culture-based methods were queried regarding future plans for adding nonculture-based methods.

Data were entered into a web-based tool, and analyses were performed with SAS v9.1 software (SAS Institute, Cary, NC). Only laboratories that performed on-site testing for STEC were included in the analysis. Culture-based methods specifically refer to performing bacterial culture on sorbitol-containing medium to identify O157 STEC. Nonculture-based methods included in the analysis were commercially available EIA kits that would detect Stx I and 2. Univariate comparisons were conducted using Fisher's two-sided exact test. We also examined the number of O157 STEC and non-O157 STEC infections reported to FoodNet in 2007 by the method used at the clinical laboratory that initially detected the case; data from Tennessee were not included in this analysis, because no identifiers were available to link each case reported to FoodNet with the initial clinical laboratory that detected that case.

A similar survey of clinical laboratories was conducted in 2003 in nine FoodNet sites (excludes New Mexico, which joined FoodNet in 2004) (CDC, unpublished data). That survey was mailed to 623 laboratories and asked similar questions regarding practices related to culture- and nonculture-based methods; 498 (86%) laboratories responded. We compared selected results from the 2007 survey with the 2003 survey results to describe changes over time.

Results

Responses to the survey were received from 664 (99%) of 672 clinical laboratories identified as serving the FoodNet catchment area. Most surveys were completed by the laboratory or microbiology supervisor (77%). Two hundred thirty-six laboratories (36%) indicated that they sent specimens to another hospital or reference laboratory and did not perform on-site testing for STEC. The remaining 428 laboratories (64%) performed on-site testing for STEC; presented results are from these laboratories.

Among the laboratories testing on site, 379 (89%) used only culture-based methods, 30 (7%) used only Stx EIA, and 19 (4%) used both Stx EIA and culture-based methods. No laboratories reported using polymerase chain reaction to identify the presence of Stx-producing genes. Among the 19 laboratories that performed both culture and Stx EIA on site, seven (37%) set up these tests simultaneously, five (26%) performed subsequent culture on all Stx-positive specimens, and seven (37%) used an algorithm not queried by our survey. Testing practices varied across the 10 FoodNet sites (Table 1).

Table 1.

Clinical Laboratory Testing Practices for Shiga Toxin–Producing Escherichia coli by Foodnet Site

Site	Number of labs testing on site	Use culture-based method only N (%)	Use Stx EIA only N (%)	Use both methods N (%)	Simultaneous use of both methods N (%)	Routinely test all stools with at least one method N (%)	Routinely test all stools with Stx EIA N (%)
California	24	23 (96)	1 (4)	0	0	13 (54)	1 (4)
Colorado	18	15 (83)	2 (11)	1 (6)	0	14 (78)	0
Connecticut	27	15 (56)	7 (26)	5 (18)	2 (7)	18 (67)	5 (19)
Georgia	60	56 (93)	3 (5)	1 (2)	0	24 (40)	4 (7)
Maryland	44	38 (86)	5 (11)	1 (3)	0	28 (64)	4 (9)
Minnesota	76	68 (89)	3 (4)	5 (7)	3 (4)	71 (93)	6 (8)
New Mexico	32	31 (97)	1 (3)	0	0	20 (63)	1 (3)
New York	51	45 (88)	4 (8)	2 (4)	1 (2)	40 (78)	3 (6)
Oregon	41	38 (93)	3 (7)	0	0	38 (92)	2 (5)
Tennessee	55	50 (91)	1 (2)	4 (7)	1 (2)	40 (73)	4 (7)
Total	428	379 (89)	30 (7)	19 (4)	7 (2)	306 (71)	30 (7)

Stx, Shiga toxin; EIA, enzyme immunoassay.

The 49 laboratories that used the Stx EIA in 2007 were asked to report the year that they initiated testing; 44 provided this information. The cumulative number of these laboratories using Stx EIA testing increased from four in 1999, with an average of five (range, two to eight) adding test availability each year. Among the 379 laboratories only performing on-site culture-based testing, 50 (13%) planned on adding a nonculture-based method to their testing protocols, 179 (48%) had no plan to add these methods, 122 (32%) were considering but had not made a final decision, and 27 (7%) were already using a nonculture-based method that would only detect O157 STEC.

Of the 398 laboratories using culture-based methods, 285 (72%) routinely did so on all stool specimens. In contrast, only 27 (55%) of the 49 laboratories using Stx EIA tested all stool specimens routinely (p = 0.03). Six of the seven laboratories that performed simultaneous culture, and Stx EIA tests did so routinely on all stool specimens.

Seventy-one percent of laboratories routinely tested all specimens with at least one method (Table 1). The remaining 122 laboratories used various criteria to initiate testing (Table 2). Among the 113 laboratories using culture-based methods, but not routinely, 77% would initiate testing on physician request and 63% when the patient had a history of bloody stool or the specimen appeared bloody. In comparison, among the 22 laboratories using Stx EIA, but not routinely, 55% would initiate testing on physician request (p = 0.03) and 27% for the same bloody stool criteria (p = 0.002). Other less frequently cited criteria for initiating testing included certain age groups, during certain seasons, and when a patient had hemolytic uremic syndrome.

Table 2.

Criteria Used to Initiate Testing by Method Among Laboratories That Did Not Test All Stool Specimens Routinely

Testing protocol	Culture-based methods (N = 113)	Stx EIA (N = 22)
On physician request^a	77%	55%
History of or visibly bloody stool^a	63%	27%
Certain age groups	9%	5%
Certain seasons (e.g., summer months)	0%	5%
Patient with hemolytic uremic syndrome	9%	9%

Columns are not mutually exclusive, because some laboratories perform both methods. More than one answer could be selected, so columns total more than 100%.

p-value significant at <0.05 compared with other method.

Among the 428 laboratories testing on site, 21 (5%) were reference laboratories. Only 10 (48%) tested all stool specimens routinely by at least one method compared with 296 (73%) of the 407 other laboratories (p = 0.03). Among the reference laboratories, 16 (76%) used only Stx EIA and 5 (24%) used only culture-based methods.

Laboratories that tested specimens using only culture-based methods reported 377 (81%) of the 465 cases of O157 STEC infection to FoodNet in 2007. Laboratories that used Stx EIA alone or in combination with culture-based methods reported 88 (19%) of the 465 cases of O157 STEC infection and 140 (87%) of the 161 cases of non-O157 STEC infection to FoodNet. The remaining cases of non-O157 STEC were detected by culture-only laboratories referring suspect stool specimens to an SPHL for further testing. Reference laboratories reported 170 (27%) STEC infections (50 [11%] O157 STEC; 120 [75%] non-O157 STEC) to FoodNet in 2007.

Most laboratories (97% of labs using culture-based methods and 98% of labs using Stx EIA) referred positive isolates or specimens to an SPHL for further testing. All reference laboratories reported sending specimens to an SPHL. Real-time submission of specimens was most common; however, 13% of the laboratories batched specimens before shipping when using Stx EIA compared with only 4% of laboratories when using culture-based methods (p = 0.06). Among the 49 laboratories performing Stx EIA, most (84%) sent the Stx-positive broth or original stool specimen to the SPHL without further testing, 22% would also send all isolates of O157 STEC obtained after culturing the broth.

Among laboratories testing on site, the proportion using the Stx EIA on at least some specimens increased from 6% in 2003 to 11% in 2007 (p = 0.005) (Table 3). The proportion using culture methods to detect O157 STEC remained about the same. Among laboratories using Stx EIA, the proportion sending a specimen, broth, or isolate to the SPHL and the proportion that routinely tested all specimens improved, albeit not a statistically significant change (83%–98%, p = 0.06 and 33%–55%, p = 0.13, respectively). Also, the proportion that routinely tested by a method that would detect O157 STEC (i.e., routinely cultured for O157 STEC or routinely used an EIA and routinely referred positive broths) only changed slightly and not significantly (66%–71%, p = 0.13).

Table 3.

Comparison of Laboratory Survey Results, Foodnet Sites, 2003 Versus 2007

	2003	2007
Among laboratories testing on site
• Used a method that would detect non-O157 STEC (Stx EIA)^a	6% (18/321)	11% (49/428)
• Used a method that would isolate STEC O157	94% (302/321)	93% (398/428)
• Routinely tested all stools with at least one method	66% (213/321)	71% (306/428)
Among laboratories testing on site and using Stx EIA
• Sent a specimen or isolate to SPHL	83% (15/18)	98% (48/49)
• Routinely tested all specimens	33% (6/18)	55% (27/49)

Comparing 2007 with 2003, p-value significant at <0.05.

STEC, Stx-producing Escherichia coli; SPHL, state public health laboratory.

Discussion

The goal of published recommendations for laboratory identification of STEC is to promote best practices across clinical laboratories to ensure that both O157 STEC and non-O157 STEC infections are identified as completely and rapidly as possible (Boyce et al., 1995; CDC, 2006, 2009; Klein et al., 2002). Reducing delays in the detection of STEC and specifically identifying O157 STEC are important for clinicians and public health professionals, making the advantages of simultaneous culture for O157 STEC and EIA screening for Stx apparent (CDC, 2009). The results of this survey are a comprehensive snapshot of clinical laboratory testing practices in the United States. In 2007, only 2% of laboratories surveyed used culture and nonculture-based tests simultaneously. An additional 1% of laboratories that reported using both methods performed culture for O157 STEC on specimens positive by EIA. Importantly, 36% of the 664 laboratories surveyed reported sending their specimens off-site to another hospital or reference laboratory for STEC testing. This practice can also delay STEC detection.

Another key component of current recommendations is to test all stools submitted for diagnosis of acute community-acquired diarrhea for the presence of STEC (CDC, 2009). At the time of this survey, more than a quarter of laboratories did not test all stool samples routinely for either O157 STEC or Stx, and this practice varied by testing method and state. Among laboratories that used on-site culture-based methods for detecting O157 STEC, most tested all stools routinely; however, fewer laboratories using Stx EIA did so routinely. Still, the percentage of laboratories using Stx EIA to routinely test all stool specimens increased compared with 2003.

Almost all laboratories surveyed reported using only one method. The use of culture-based methods alone means that infections caused by non-O157 STEC are rarely detected. Studies have found that non-O157 STEC may account for 28%–52% of all clinically detected STEC infections (DuPont, 2009; Klein et al., 2006; Klein et al., 2002). The vast majority of non-O157 STEC strains were reported almost exclusively by the small proportion of laboratories using Stx EIA. This suggests that laboratories not using Stx EIA are under-identifying non-O157 STEC infections. Although screening stool specimens for Stx using EIA is a faster approach for many clinical laboratories and detects more strains of STEC than culture-based methods, it is important that EIA methods do not replace culture. The use of the Stx EIA without simultaneous or prompt subsequent culture delays subtyping of isolates and does not provide the physician with confirmation or rule out infection with O157 STEC. O157 STEC is responsible for most cases of severe disease (Banatvala et al., 2001); therefore, a finding indicating the presence of O157 STEC within 24 hours after the specimen is submitted to the laboratory helps physicians rapidly gauge the risk of severe disease for the patient and for contacts and assess the need to initiate measures to prevent kidney damage and death (Ake et al., 2005; Tarr, 2009). Studies have also found that EIAs failed to detect a subset of O157 STEC which were readily identified on simultaneously plated SMAC agar, underscoring the importance of primary isolation (Fey et al., 2000; Klein et al., 2002; Manning et al., 2007). The sensitivity and positive predictive value of EIA products can vary by manufacturer and by characteristics of the population tested and may produce false positive results, making cultures of positive broths (or the original specimen) important (CDC, 2000, 2006; Gavin et al., 2004; Kehl et al., 1997).

Laboratories not located in, operated by, or affiliated with a particular hospital or hospital systems are often termed “reference or referral laboratories”. These laboratories typically handle larger specimen volumes and can accommodate the testing requirements of expansive geographic regions. Therefore, the testing practices at these laboratories can have a huge impact on both patient care and public health surveillance. Despite representing only 5% of all the laboratories that tested on-site in 2007, reference laboratories detected more than a quarter of STEC cases. None of the reference laboratories used more than one method, and fewer than half tested all stool specimens routinely. It is clearly important that the message of best practices for timely identification of STEC should reach these laboratories.

One recommendation almost universally practiced was submission of positive specimens to an SPHL for further testing, and this had improved compared with 2003. Clinical laboratories have long been encouraged or required to forward O157 STEC isolates to their SPHL as part of routine public health surveillance (Voetsch et al., 2004). However, delays can result when laboratories batch specimens before shipping.

At the SPHLs, the presence of Stx in broth can be confirmed and STEC isolated and often serogrouped (serotyping of uncommon strains is done at the CDC). The molecular subtyping of STEC isolates using pulsed-field gel electrophoresis as part of the CDC PulseNet program is another powerful tool in the surveillance and epidemiological characterization of strains (Swaminathan et al., 2001). This is an important step in the detection and investigation of O157 STEC outbreaks and, thus, in controlling the spread of infection; delays in subtyping can delay identification and control of outbreaks.

One limitation of the present analysis was that data were not collected on the numbers of stool specimens processed by each laboratory so that rates of isolation could be calculated and compared with laboratory protocols and disease incidence. Past studies have found it difficult to obtain accurate estimates (Voetsch et al., 2004). An additional limitation is that clinical laboratories practices may have changed since the time this survey was conducted; however, this study presents the most recent and diverse estimate of clinical laboratory practices in the United States. In future surveys, it may be prudent to query laboratories about the reasons for their decisions regarding in-house testing protocols (such as staffing and cost efficiency, public health importance) and how much national guidelines influence these decisions.

Screening of stool specimens by clinical laboratories for Stx using nonculture-based methods, simultaneous or subsequent culturing to isolate O157 STEC, and forwarding O157 STEC isolates and enrichment broths from Stx-positive specimens that do not yield O157 to SPHLs are vital steps for public health surveillance of STEC infections. Most clinical laboratories in FoodNet sites did not conduct Stx EIA testing; even fewer did so routinely. Simultaneous culture and EIA testing were rare. Efforts are being made to encourage this type of coordinated approach at clinical laboratories, including the release of comprehensive guidelines for STEC testing by clinical laboratories in 2009 (CDC, 2009).

Future laboratory surveys should assess the impact of the 2009 guidelines. It could also be useful to query laboratories about the reasons for their decisions regarding in-house testing protocols (such as staffing and cost efficiency, public health importance) and how much national guidelines may influence such decisions. Accurate and timely laboratory testing for STEC will ensure the prompt diagnosis and treatment of these infections and when combined with epidemiological information will help improve surveillance and detection of outbreaks.

Footnotes

Acknowledgments

We thank all of the FoodNet surveillance officers who conducted the survey. Financial support: Funding for this report was provided by the Centers for Disease Control and Prevention, the United States Department of Agriculture, and the United States Food and Drug Administration. The findings and conclusions in this report are those of the authors and do not necessarily represent those of the funding agencies.

Disclosure Statement

No competing financial interests exist.

References

Ake

, Jelacic

, Ciol

et al. Relative nephroprotection during Escherichia coli O157:H7 infections: association with intravenous volume expansion. Pediatrics, 2005; 115:e673–e680.

Andreoli

, Trachtman

, Acheson

DWK

et al. Hemolytic uremic syndrome: epidemiology, pathophysiology, and therapy. Pediatr Nephrol, 2002; 17:293–298.

Angulo

, Voetsch

, Vugia

et al.

Determining the burden of human illness from food borne diseases: CDC's Emerging Infectious Disease Program Foodborne Diseases Active Surveillance Network (FoodNet)

Vet Clin North Am Food Anim Pract, 1998; 14:165–172.

Banatvala

, Griffin

, Greene

et al. The United States national prospective hemolytic uremic syndrome study: microbiologic, serologic, clinical, and epidemiologic findings. J Infect Dis, 2001; 183:1063–1070.

Besser

, Griffin

. Escherichia coli O157:H7 gastroenteritis and the hemolytic uremic syndrome: an emerging infectious disease. Annu Rev Med, 1999; 50:355–367.

Boyce

, Pemberton

, Wells

, Griffin

. Screening for Escherichia coli O157:H7- a nationwide survey of clinical laboratories. J Clin Microbiol, 1995; 33:3275–3277.

Brooks

, Sowers

, Wells

et al. Non-O157 Shiga toxin-producing Escherichia coli infections in the United States, 1983–2002. J Infect Dis, 2005; 192:1422–1429.

[CDC] U.S. Centers for Disease Control and Prevention. Importance of culture confirmation of Shiga toxin-producing Escherichia coli infection as illustrated by outbreaks of gastroenteritis—New York and North Carolina. MMWR Morb Mortal Wkly Rep, 2006; 55:1042–1045.

[CDC] U.S. Centers for Disease Control and Prevention. Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food—10 states 2007. MMWR Morb Mortal Wkly Rep, 2008; 57:366–370.

10.

[CDC] U.S. Centers for Disease Control and Prevention. Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food—10 states, 2008. MMWR Morb Mortal Wkly Rep, 2009; 58:333–337.

11.

[CDC] U.S. Centers for Disease Control and Prevention. Recommendations for diagnosis of Shiga toxin-producing Escherichia coli infections by clinical laboratories. MMWR, 2009; 58,RR-12:1–12.

12.

[CDC] U.S. Centers for Disease Control and Prevention. University outbreak of Calicivirus infection mistakenly attributed to Shiga toxin-producing Escherichia coli O157:H7—Virginia. MMWR Morb Mortal Wkly Rep, 2000; 50:489–491.

13.

Cohen

, Jones

, Schermond

, Gerber

. Identifying Shiga toxin-producing Escherichia coli: results of a laboratory evaluation. Foodborne Path Dis, 2008; 5:295–301.

14.

DuPont

. Bacterial diarrhea. New Engl J Med, 2009; 361:1560–1569.

15.

Fey

, Wickert

, Rupp

et al. Prevalence of non-O157:H7 Shiga toxin–producing Escherichia coli in diarrheal stool samples from Nebraska. Emerg Infect Dis, 2000; 6:530–533.

16.

Gavin

, Peterson

, Pasquariello

et al. Evaluation of performance and potential clinical impact of ProSpecT Shiga toxin Escherichia coli microplate assay for detection of Shiga toxin-producing E. coli in stool samples. J Clin Microbiol, 2004; 42:1652–1656.

17.

Johnson

, Thorpe

, Sears

. The emerging clinical importance of non-O157 Shiga toxin-producing Escherichia coli. Clin Infect Dis, 2006; 43:1587–1595.

18.

Kehl

, Havens

, Behnke

, Acheson

DWK

. Evaluation of the Premier EHEC assay for detection of Shiga toxin-producing Escherichia coli. J Clin Microbiol, 1997; 35:2051–2054.

19.

Klein

, Boster

, Stapp

et al. Diarrhea etiology in a Children's Hospital Emergency Department: a prospective cohort study. Clin Infect Dis, 2006; 43:807–813.

20.

Klein

, Stapp

, Clausen

et al. Shiga toxin-producing Escherichia coli in children with diarrhea: a prospective point-of-care study. J Pediatrics, 2002; 141:172–177.

21.

Manning

, Madera

, Schneider

et al. Surveillance for Shiga toxin–producing Escherichia coli, Michigan, 2001–2005. Emerg Infect Dis, 2007; 13:318–321.

22.

Persson

, Olsen

, Ethelberg

, Scheutz

. Subtyping method for Escherichia coli Shiga toxin (Verocytotoxin) 2 variants and correlations to clinical manifestations. J Clin Microbiol, 2007; 45:2020–2024.

23.

Swaminathan

, Barrett

, Hunter

et al. PulseNet: the molecular subtyping network for foodborne bacterial disease surveillance, United States. Emerg Infect Dis, 2001; 7:382–389.

24.

Tarr

. Shiga toxin-associated hemolytic uremic syndrome and thrombotic thrombocytopenic purpura: distance mechanims of pathogenesis. Kidney Int Suppl, 2009; 112:S29–S32.

25.

Voetsch

, Angulo

, Rabatsky-Her

et al. Laboratory practices for stool-specimen culture for bacterial pathogens, including Escherichia coli O157:H7, in the FoodNet sites, 1995–2000. Clin Infect Dis, 2004; 38:S190–S197.