Recommendations for Diagnosis of Shiga Toxin--Producing Escherichia coli Infections by Clinical Laboratories

Recommendations for Diagnosis of Shiga Toxin--Producing Escherichia coli Infections by Clinical Laboratories
Prepared by
L. Hannah Gould1
Cheryl Bopp1
Nancy Strockbine1
Robyn Atkinson2
Vickie Baselski3,4
Barbara Body5
Roberta Carey6
Claudia Crandall7
Sharon Hurd8
Ray Kaplan9
Marguerite Neill10
Shari Shea11
Patricia Somsel12
Melissa Tobin-D'Angelo13
Patricia M. Griffin1
Peter Gerner-Smidt1

1Division of Foodborne, Bacterial, and Mycotic Diseases, National Center for Zoonotic, Vector-Borne, and Enteric Diseases, CDC, Atlanta, Georgia

2Knoxville Regional Laboratory, Tennessee Department of Health, Knoxville, Tennessee

3American Society for Microbiology, Washington, DC

4University of Tennessee Health Science Center, Memphis, Tennessee

5LabCorp, Burlington, North Carolina

6Division of Healthcare Quality Promotion, National Center for Preparedness, Detection, and Control of Infectious Diseases, CDC, Atlanta, Georgia

7California Department of Public Health, Richmond, California

8Connecticut Emerging Infections Program, New Haven, Connecticut

9Quest Diagnostics, Tucker, Georgia

10Brown University, Warren Alpert School of Medicine, Providence, Rhode Island

11Association of Public Health Laboratories, Silver Spring, Maryland

12Michigan Department of Community Health, Lansing, Michigan

13Georgia Division of Public Health, Atlanta, Georgia

Corresponding preparer: L. Hannah Gould, Division of Foodborne, Bacterial, and Mycotic Diseases, National Center for Zoonotic, Vector-Borne, and Enteric Diseases, CDC, 1600 Clifton Road, N.E., MS A-38, Atlanta, GA 30333. Telephone: 404-639-3373; Fax: 404-639-3535; E-mail: lgould@cdc.gov.

Summary
Shiga toxin--producing Escherichia coli (STEC) are a leading cause of bacterial enteric infections in the United States. Prompt, accurate diagnosis of STEC infection is important because appropriate treatment early in the course of infection might decrease the risk for serious complications such as renal damage and improve overall patient outcome. In addition, prompt laboratory identification of STEC strains is essential for detecting new and emerging serotypes, for effective and timely outbreak responses and control measures, and for monitoring trends in disease epidemiology. Guidelines for laboratory identification of STEC infections by clinical laboratories were published in 2006 (1). This report provides comprehensive and detailed recommendations for STEC testing by clinical laboratories, including the recommendation that all stools submitted for routine testing from patients with acute community-acquired diarrhea (regardless of patient age, season of the year, or presence or absence of blood in the stool) be simultaneously cultured for E. coli O157:H7 (O157 STEC) and tested with an assay that detects Shiga toxins to detect non-O157 STEC. The report also includes detailed procedures for specimen selection, handling, and transport; a review of culture and nonculture tests for STEC detection; and clinical considerations and recommendations for management of patients with STEC infection. Improving the diagnostic accuracy of STEC infection by clinical laboratories should ensure prompt diagnosis and treatment of these infections in patients and increase detection of STEC outbreaks in the community.

Introduction
Shiga toxin--producing E. coli (STEC) cause approximately 100,000 illnesses, 3,000 hospitalizations, and 90 deaths annually in the United States, according to the last estimate in 1999 (2). Most reported STEC infections in the United States are caused by E. coli O157:H7, with an estimated 73,000 cases occurring each year (2). Non-O157 STEC bacteria also are important causes of diarrheal illness in the United States; at least 150 STEC serotypes have been associated with outbreaks and sporadic illness (2--4). In the United States, six non-O157 serogroups (O26, O45, O103, O111, O121, and O145) account for the majority of reported non-O157 STEC infections (5).

The toxins produced by STEC were named based on their similarity in structure and function to Shiga toxins produced by Shigella dystenteriae type 1 (6). Shiga toxin 1 (Stx1) is neutralized by antibodies against Shiga toxin, whereas Shiga toxin 2 (Stx2) is not neutralized by antibodies against Shiga toxin but is neutralized by homologous antibodies. STEC are also referred to as verocytotoxigenic E. coli; STEC that cause human illness are also referred to as enterohemorrhagic E. coli. In this report, all E. coli that produce a Shiga toxin are referred to as STEC. STEC serotypes are named according to their somatic (O) and flagellar (H) antigens. In this report, all STEC with the O antigen 157 are referred to as O157 STEC, regardless of whether the H7 antigen has been identified or Shiga toxin production has been confirmed. STEC with other O antigens are referred to as non-O157 STEC or by their specific O antigen.

STEC infection causes acute, often bloody, diarrhea. Approximately 8% of persons who receive a diagnosis of O157 STEC infection develop hemolytic uremic syndrome (HUS), a life-threatening condition characterized by thrombocytopenia, hemolytic anemia, and renal failure (7--9). Thrombotic thrombocytopenic purpura (TTP), a syndrome with signs and symptoms that are similar to those of HUS, is typically diagnosed in adults. When TTP is diagnosed after a diarrheal illness, the condition is usually caused by infection with O157 STEC or another STEC. In this report, regardless of the age of the patient, TTP diagnosed after a diarrheal illness is referred to as HUS (10).

Whether an illness progresses to HUS depends on strain virulence and host factors (11). Although most persons with diarrhea-associated HUS have an O157 STEC infection, certain non-O157 STEC strains also can lead to HUS (3). The virulence of non-O157 STEC is partly determined by the toxins they produce; non-O157 STEC strains that produce only Stx2 are more often associated with HUS than strains that produce only Stx1 or that produce both Stx1 and Stx2 (12). STEC infections and HUS occur in persons of all ages, but the incidence of STEC infection is highest in children aged <5 years, as is the risk for HUS (9). Although STEC infections are more common during summer months, they can occur throughout the year.

STEC transmission occurs through consumption of a wide variety of contaminated foods, including undercooked ground beef, unpasteurized juice, raw milk, and raw produce (e.g., lettuce, spinach, and alfalfa sprouts); through ingestion of contaminated water; through contact with animals or their environment; and directly from person to person (e.g., in child-care settings). Both O157 STEC and O111 STEC have a low infectious dose (<100 organisms) (13); the infectious dose of other serogroups is not known.

Prompt and accurate diagnosis of STEC infection is important because appropriate treatment with parenteral volume expansion early in the course of infection might decrease renal damage and improve patient outcome (14). In addition, because antibiotic therapy in patients with STEC infections might be associated with more severe disease, prompt diagnosis is needed to ensure proper treatment. Furthermore, prompt laboratory identification of STEC strains is essential for implementation of control measures, for effective and timely outbreak responses, to detect new and emerging serotypes, and to monitor trends in disease epidemiology (1,15,16).

Most O157 STEC isolates can be readily identified in the laboratory when grown on sorbitol-containing selective media because O157 STEC cannot ferment sorbitol within 24 hours. However, many clinical laboratories do not routinely culture stool specimens for O157 STEC. In addition, selective and differential media are not available for the culture of non-O157 STEC, and even fewer laboratories culture stool specimens for these bacteria than for O157 STEC.

Recently, the increased use of enzyme immunoassay (EIA) or polymerase chain reaction (PCR) to detect Shiga toxin or the genes that encode the toxins (stx1 and stx2) has facilitated the diagnosis of both O157 and non-O157 STEC infections. Although EIA and other nonculture tests are useful tools for diagnosing STEC infection, they should not replace culture; a pure culture of the pathogen obtained by the clinical laboratory (O157 STEC) or the public health laboratory (non-O157 STEC) is needed for serotyping and molecular characterization (e.g., pulsed-field gel electrophoresis [PFGE] patterns), which are essential for detecting, investigating, and controlling STEC outbreaks.

Simultaneous culture of stool for O157 STEC and EIA testing for Shiga toxin is more effective for identifying STEC infections than the use of either technique alone (17,18). Because virtually all O157 STEC have the genes for Stx2 (stx2) and intimin (eae), which are found in strains that are associated with severe disease (5,12,19--22), detection of O157 STEC should prompt immediate initiation of steps such as parenteral volume expansion to reduce the risk for renal damage in the patient and the spread of infection to others.

Guidelines for clinical and laboratory identification of STEC infections have been previously published (1); this report provides the first comprehensive and detailed recommendations for isolation and identification of STEC by clinical laboratories. The recommendations are intended primarily for clinical laboratories but also are an important reference for health-care providers, public health laboratories, public health authorities, and patients and their advocates.

Recommendation for Identification of STEC by Clinical Laboratories
All stools submitted for testing from patients with acute community-acquired diarrhea (i.e., for detection of the enteric pathogens Salmonella, Shigella, and Campylobacter) should be cultured for O157 STEC on selective and differential agar. These stools should be simultaneously assayed for non-O157 STEC with a test that detects the Shiga toxins or the genes encoding these toxins. All O157 STEC isolates should be forwarded as soon as possible to a state or local public health laboratory for confirmation and additional molecular characterization (i.e., PFGE analysis and virulence gene characterization). Detection of STEC or Shiga toxin should be reported promptly to the treating physician, to the public health laboratory for confirmation, isolation, and subsequent testing of the organism, and to the appropriate public health authorities for case investigation. Specimens or enrichment broths in which Shiga toxin or STEC are detected but from which O157 STEC are not recovered should be forwarded as soon as possible to a state or local public health laboratory.

Benefits of Recommended Testing Strategy
Identification of Additional STEC Infections and Detection of All STEC Serotypes
Evidence indicates that STEC might be detected as frequently as other bacterial pathogens. In U.S. studies, STEC were detected in 0%--4.1% of stools submitted for testing at clinical laboratories, rates similar to those of Salmonella species (1.9%--4.8%), Shigella species (0.2%--3.1%), and Campylobacter species (0.9%--9.3%) (9,17,23--31). In one study, the proportion of stools with STEC detected varied by study site (9); O157 STEC were more commonly isolated than some other enteric pathogens in northern states. The laboratory strategy of culturing stool while simultaneously testing for Shiga toxin is more sensitive than other strategies for STEC identification and ensures that all STEC serotypes will be detected (17,18,30,31) (Table 1). In addition, immediate culture ensures that O157 STEC bacteria are detected within 24 hours of the initiation of testing.

Early Diagnosis and Improved Patient Outcome
Early diagnosis of STEC infection is important for determining the proper treatment promptly. Initiation of parenteral volume expansion early in the course of O157 STEC infection might decrease renal damage and improve patient outcome (14). Conversely, certain treatments can worsen patient outcomes; for example, antibiotics might increase the risk for HUS in patients infected with O157 STEC, and antidiarrheal medications might worsen the illness (32). Early diagnosis of STEC infection also might prevent unnecessary procedures or treatments (e.g., surgery or corticosteroids for patients with severe abdominal pain or bloody diarrhea) (33--35).

Prompt Detection of Outbreaks
Prompt laboratory diagnosis of STEC infection facilitates rapid subtyping of STEC isolates by public health laboratories and submission of PFGE patterns to PulseNet, the national molecular subtyping network for foodborne disease surveillance (36). Rapid laboratory diagnosis and subtyping of STEC isolates leads to prompt detection of outbreaks, timely public health actions, and detection of emerging STEC strains (37,38). Delayed diagnosis of STEC infections might lead to secondary transmission in homes, child-care settings, nursing homes, and food service establishments (39,40--44) and might delay detection of multistate outbreaks related to widely distributed foods (39,45). Outbreaks caused by STEC with multiple serogroups (46) or PFGE patterns (47) have been documented.

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