Recommendations for Collection of Laboratory Specimens Associated with Outbreaks of Gastroenteritis

This report was prepared by: Judy F. Lew, MD Charles W. LeBaron, MD Roger I. Glass, MD Tom Torok, MD Patricia M. Griffin, MD Joy G. Wells, MS Dennis D. Juranek, DVM Suzanne P. Wahlquist, MS

Summary

Recent discoveries have implicated a number of "new" (i.e., previously unrecognized) infectious agents as important causes of outbreaks of gastroenteritis. Unfortunately, the ability to detect these agents in an outbreak can be limited by two factors:

1. the lack of appropriate assays--many of which are still in developmental stages and are not readily available to clinical laboratories, and 2) inadequately or improperly collected specimens. At CDC, many newly developed assays are being used for research and for outbreak investigations. The information in this report is especially intended for public health agencies that collaborate with CDC in investigating outbreaks of gastroenteritis. The report provides an update on guidelines and recommendations for the proper collection of specimens to be sent to CDC, gives general background information concerning some recently discovered pathogens, lists some of the tests available at CDC, and provides a list of CDC contacts.* The guidelines and the general information provided on causes of outbreaks of gastroenteritis can be also used by public health workers for investigations when specific testing is available and appropriate. I.INTRODUCTION

New diagnostic laboratory techniques as well as modifications of standard ones have been used by investigators to identify viral, bacterial, and parasitic agents of outbreaks of gastroenteritis. Recently discovered causative agents associated with such outbreaks include the Norwalk-like agents, astrovirus, calicivirus, enteric adenovirus, enterohemorrhagic Escherichia coli, Cryptosporidium, and Isospora belli. Use of direct electron microscopy (EM), coupled with immunologic techniques (e.g., immune EM and enzyme immunoassays (EIAs)) and serologic studies, has enhanced understanding of viruses as a major cause of gastroenteritis. Bacteria and parasites continue to be recognized as important causes of diarrhea worldwide. Use of EIAs, tissue culture, molecular probes, and the polymerase chain reaction has improved the diagnosis of diarrhea caused by bacteria, and special concentrating and staining techniques have improved the process of detecting parasites such as Cryptosporidium and I. belli.

To ensure that these diagnostic advances can be used most effectively, earlier recommendations for collecting stool specimens must be updated. Because appropriate specimen-collection methods for viral, bacterial, and parasitic agents differ, the sections below are divided into guidelines by category of agent. II. PROCEDURES FOR COLLECTING SPECIMENS

1. Stool (See Table 1)

1. General guidelines

1. Timing. Begin collecting stool specimens immediately

after being notified of an outbreak, since delay may impede identification of the causative agent. To permit diagnosis of certain viral agents, specimens must be collected during the first 48 hours of illness.

b. Quantity. Collect diarrheal stool samples from at least 10 ill persons (assuming that at least that number are involved in the outbreak). For outbreaks thought to be of viral origin, collect large-volume stool specimens (at least a urine cupful).

2. Methods for collection and storage

1. Viruses. Place each diarrheal stool specimen, of as

large a quantity as can be obtained (preferably, at least 10 cc), in a leak-proof, clean, dry container, and refrigerate at 4 C. Instructing patients to catch stool specimens in plastic kitchen wrap draped across the back half of the toilet under the toilet seat may facilitate collection of stool specimens. Do not freeze specimens if EM examination is anticipated.

b. Bacteria. Collect at least two rectal swabs or swabs of fresh stools from each patient and place swabs in refrigerated (i.e., chilled 1-2 hours before use) Cary-Blair transport medium. When obtaining swabs from a patient, first moisten each rectal swab in the holding medium, insert the moistened swab into the rectum 1 to 1-1/2 inches, rotate the swab gently, and then return the swab to the same tube of holding medium. Try to ensure that visible fecal material is present on each swab. After obtaining the two fecal swabs, insert both into the same tube of medium and push them to the bottom of the tube. Break off and discard the excess top portion of the swab sticks.

IMPORTANT: Refrigerate or freeze tubes after specimens are placed in them. If specimens will be examined within 48 hours after collection, they can be refrigerated; however, if specimens must be held longer than 48 hours, freeze them as soon as possible after they are collected. Although storage in an ultra-low freezer (-70 C) is preferable, storage in a home-type freezer (if it is properly set at -20 C) is acceptable for short periods.

c. Parasites. Mix fresh bulk-stool specimens thoroughly with each of two preservatives, 10% formalin and polyvinyl alcohol (PVA) fixative,* at a ratio of 1 part stool to 3 parts preservative. If there is a delay in obtaining the preservatives, refrigerate untreated stool specimens at 4 C (do not freeze) for up to 48 hours. Once preserved, the specimens can be stored and transported at room temperature or refrigerated. Do not freeze.

3. Transportation (specimen handling)

1. Refrigerated specimens. Enclose each specimen in a

secure container (e.g., urine cup, Cary-Blair medium tube), to which has been affixed a waterproof label. Place this container in a waterproof bag with tissue, towels, or other blotting material to absorb any leakage. Batch specimen containers, pack with ice or frozen refrigerant packs in an insulated box, and send by overnight mail scheduled to be delivered during business hours on a weekday, if possible. Submit a list of specimens and a CDC Identification Form 50.34 for each specimen.

b. Frozen specimens (for bacterial testing only). So that they remain frozen, ship frozen specimens on dry ice. Use enough dry ice to keep the specimen frozen until it is received at the laboratory that will process it (i.e., enough dry ice to fill one-third to one-half of the shipping container). Do not allow glass tubes to be in direct contact with dry ice; place a layer of paper or other material between the tubes and the dry ice. To prevent excess exposure of specimens to carbon dioxide, tighten the screw caps on the Cary-Blair tubes and seal them with electrical tape, or seal the specimens in a plastic bag within the container of dry ice.

B. Serum

1. General guidelines

1. Timing of collection of specimens. Submit two serum

specimens (an acute-phase specimen and a convalescent-phase specimen) for each patient thought to have illness caused by viruses or bacteria. Obtain the acute-phase serum specimen as close to the time of onset of illness as possible (at most, within a week after onset of illness) and the convalescent-phase serum specimen 3-4weeks after the onset of illness. If a viral agent is suspected, for optimal test results, specimens should be collected within 6 weeks after onset of illness.

b. Source(s) of specimens. If possible, obtain paired serum specimens from the same 10 patients from whom stool samples were obtained. Ten paired serum specimens obtained from well persons can serve as control specimens in certain studies.

2. Methods for collection and storage

Collect blood specimens from adults (15 ml) and from children (3 ml) in tubes that do not contain anticoagulants (usually red-top tubes). Centrifuge the blood and send only the serum for analysis. If no centrifuge is available, store the blood specimens in a refrigerator until a clot has formed; then remove the serum and pipette it into an empty sterile tube (using a Pasteur pipette). Refrigerate the tubes of spun serum until they are shipped. Refrigerate, but do not freeze, tubes containing unspun serum.

3. Transportation

Ship serum specimens either refrigerated or frozen. If the clotting technique described above is used to obtain the serum, ship the specimens refrigerated so that they can be centrifuged before they are frozen. Specimens can be refrigerated by placing them in an insulated box with ice or frozen refrigerant packs. Frozen specimens can be kept frozen by shipping them on dry ice. Batch the specimens and send by overnight mail, scheduled to arrive at the laboratory during business hours on a weekday, if possible. NOTE: Certain specimens of food, water, or ice can be submitted for testing if they are epidemiologically implicated as the vehicle of the disease. However, because of the technical difficulties and the low yield associated with such studies, only send such specimens to CDC after staff in the appropriate laboratory have agreed. Refrigerate specimens for viral and parasitic diagnostic testing at 4 C, and freeze specimens for bacterial diagnostics at -70 C. III. PATHOGENS OF DIARRHEA

1. Viruses In the 1970s, specific viruses were first identified as infectious agents of diarrhea when transmission EM was used to detect viruses directly in fecal specimens. Agents such as group A rotavirus and Norwalk virus are now well-known causes of gastroenteritis (See Table 2). However, little is known about many other viral agents, including the group B and C rotaviruses, Norwalk-like agents (i.e., small, round, structured viruses), astroviruses, caliciviruses, enteric adenoviruses, pestiviruses, enteric coronaviruses, and picobirnaviruses. Group B rotavirus has been associated with large outbreaks of diarrhea among adults in China, but little is known about its effect in other parts of the world. Group C rotavirus has been reported worldwide in association with sporadic diarrhea and with outbreaks of diarrheal illness in the United Kingdom and Japan. The Norwalk-like family of viruses has been associated with disease both among adults and school-age children. Astroviruses and caliciviruses have also been implicated in outbreaks of diarrhea affecting both children and adults, particularly in such closed community settings as hospitals, orphanages, and nursing homes. Enteric adenoviruses can cause infections among immunocompromised patients, and studies suggest that these agents may be the second most common viral cause (after rotavirus) of diarrheal illness that results in the hospitalization of children.

These viruses are most likely spread by fecal-oral transmission. However, shellfish (e.g., oysters, clams, and cockles) have been documented as vehicles for outbreaks of gastroenteritis caused by Norwalk-like agents, astrovirus, and calicivirus. Most patients shed these viruses in the greatest amounts during the acute phase of illness and up to 48-72 hours post-recovery. Patients have been reported to shed enteric adenoviruses for a longer period after recovery, and immunocompromised hosts have been noted to shed viruses for months. Airborne transmission has been suggested for some members of the Norwalk family of viruses and for rotavirus. Direct zoonotic transmission has not been reported.

Outbreaks of viral gastroenteritis are characterized by a short incubation period: 24-48 hours when the agent is one of the Norwalk family of agents or a rotavirus and 24-96 hours when the agent is an astrovirus or a calicivirus. Enteric infection with adenovirus can involve a longer incubation period--i.e., 7-8 days. Non-bloody diarrhea, vomiting, abdominal cramps, and fever are common symptoms of viral gastroenteritis. Vomiting can be pronounced and can occur with or without diarrhea. Symptoms generally last for 2-5 days, although the duration of diarrhea associated with enteric adenoviruses is usually longer (7-10 days). The epidemiology of these agents has not yet been well-defined. The development and application of new immunoassays should provide new information about these agents and promote the development of effective prevention and treatment regimens. B. Bacteria

Many different bacteria cause outbreaks of diarrhea (See Table 3). Staphylococcus aureus and Clostridium perfringens are common pathogens that cause illness when their toxin is ingested. Other common pathogens include Campylobacter, Salmonella, and Shigella. Although E. coli has long been known to cause diarrhea, the recent identification of new strains suggests that this pathogen may be a more important cause of diarrheal illness than was previously recognized. Diarrheagenic E. coli organisms are currently categorized into four groups: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), and enterohemorrhagic E. coli (EHEC). ETEC and EPEC are important causes of diarrhea for infants and children in developing countries. Neither group of E. coli is often associated with outbreaks of diarrhea in the United States, although EPEC was responsible for nursery-based epidemics of diarrhea that resulted in high mortality in this country in the 1940s. ETEC remains an important cause of travelers' diarrhea. EIEC has also been identified in the United States only rarely. In contrast, EHEC (of which E. coli 0157:H7 is the prototype) is frequently reported in this country. E. coli 0157:H7 was first recognized as a cause of human illness during an outbreak of hemorrhagic colitis in 1982, and it is now known to be the major cause of hemolytic uremic syndrome in the United States. Cattle appear to be a common reservoir for this organism, and outbreaks have been traced to ground beef and raw milk.

The typical clinical manifestations of diarrheagenic E. coli are profuse, watery, cholera-like diarrhea (ETEC), dysentery-like symptoms (EIEC), and grossly bloody diarrhea (EHEC). Infection with EPEC is characterized by prolonged, relapsing episodes of watery diarrhea, although its clinical features, like those of the other diarrheagenic E. coli, can vary greatly. E. coli-related illness typically lasts longer than most diarrhea caused by viruses, and 5-7 days of illness is not unusual. Identification of these E. coli organisms can require specialized laboratory techniques such as serotyping and specific toxin-detection tests that may not be routinely available in hospital laboratories. C. Parasites

Entamoeba histolytica, Giardia lamblia, Balantidium coli, Cryptosporidium, and Isospora belli are parasitic diarrheal pathogens with a worldwide distribution. All five pathogens are known to cause outbreaks of diarrhea in the United States (See Table 4). With the use of new concentrating and staining techniques, investigators have recently shown Cryptosporidium and

1. belli to be important pathogens in the United States. Cryptosporidium has been found in specimens from 3% to 13% of persons with diarrhea who have recently arrived from developing countries. Because Cryptosporiduim has been associated with outbreaks of diarrhea in child day-care centers, it may be an important pathogen in this setting. Cryptosporidium, like I. belli, is a common source of infection for male homosexuals. I. belli appears to be a less common cause of diarrhea, although it can cause severe diarrhea for immunocompromised patients. I. belli has also been associated with outbreaks of diarrhea in some developing countries and in nursing homes in developed countries.

Fecal-oral spread through contaminated food and water has led to outbreaks of diarrhea caused by Giardia lamblia, Entamoeba histolytica, and Cryptosporidium. Person-to-person transmission has been documented for Giardia lamblia, Entamoeba histolytica,

1. belli, and Cryptosporidium. Cryptosporidium is a zoonotic agent, and many different animals are known to excrete Cryptosporidium cysts that can potentially infect humans.

1. belli is not a zoonotic agent; it is the only Isospora

species that is presently known to infect humans.

In general, the incubation period, duration of illness, and period of pathogen excretion are usually longer in association with diarrheal infections caused by parasites than with diarrhea caused by viruses or bacteria. For most patients, the incubation period for Cryptosporidium-related illness is approximately 5 days. Symptoms include transient diarrhea with profuse watery stools, abdominal cramps, and occasional fever and nausea with or without vomiting. The duration of illness is usually 3 days to 3 weeks, but illness has been documented to last for greater than 1 month.

For immunocompromised patients, clinical features can be quite marked; the illness can become progressively more severe and can include signs of malabsorption and of fluid and electrolyte loss. Although less is known about the clinical manifestations of illness caused by I. belli, current information suggests that the illness it causes is similar to that caused by Cryptosporidium. IV. DETECTION OF PATHOGENS*

1. Viruses

1. General guidelines Some of the tests presently available are direct EM;

immune EM; polyacrylamide gel electrophoresis; enzyme immune assays for rotavirus (groups A and B), enteric adenovirus, and astrovirus antigens; a serologic enzyme immune assay for Norwalk virus; and polymerase chain reaction for rotavirus serotyping. EIAs for calicivirus antigens and EIAs for astrovirus antibodies are being developed.

1. Stool. Many viruses--including rotaviruses,

adenoviruses, astroviruses, caliciviruses, the Norwalk virus, and other small, round viruses--can be detected with EM. Because the electron microscope scans a field containing a millionth of a milliliter of stool, the specimen must contain at least a million particles per milliliter for one particle of virus to be detected. In order to assure that samples contain the highest concentration of virus possible, specimens must be collected as close to the onset of illness as possible, generally within the first 48-72 hours. Watery stool specimens are most likely to contain visible particles of virus.

Freezing may alter or obliterate the morphologic characteristics of some viruses; therefore, samples should be kept refrigerated at 4 C and should not be frozen if EM examination is anticipated. Because most enteric viruses (e.g., calicivirus, the Norwalk agents, and the non-group A rotaviruses) cannot be cultivated, reagents for diagnostic tests are limited. In fact, because human stool samples are the basic source of antigen and virus, the bulk collection of specimens is always encouraged. Proper collection and storage of large-volume specimens will facilitate EM examination and allow for concentration methods that enhance the likelihood of successful testing.

b. Serum.

Antibodies to viruses usually begin to rise the first week after onset of illness, peak by the fourth week, and can fall by the sixth week (particularly for Norwalk agents). Acute-phase serum specimens should be collected in the first week of illness, and convalescent-phase serum specimens from the third to the fourth week after onset of illness (not later than the sixth week if the suspected pathogen is a Norwalk agent). Since many persons have preexisting antibodies to some of the viral agents, a single convalescent-phase serum sample is generally of little diagnostic value. At present, determination of the cause of outbreaks of viral gastroenteritis relies heavily on assays of serologic response; paired serum specimens should therefore be submitted for testing associated with all such outbreaks. A fourfold rise in specific antibody titer between acute-and convalescent-phase serum samples is accepted as diagnostic of a recent infection. B. Bacteria

1. General guidelines Tests available for identification of organisms include

isolation procedures for Salmonella, Shigella, Campylobactor, Vibrio, Yersinia enterocolitica, Aeromonas hydrophilia, Plesiomonas shigelloides, Bacillus cereus, Staphylococcus aureus, and Escherichia coli, as well as serodiagnostic assays for Salmonella typhi, Campylobacter, Vibrio cholerae, and E. coli 0157:H7. When no other pathogen is found, serotyping of E. coli recovered from ill persons and from appropriately chosen controls can identify previously unidentified E. coli pathogens. In addition, polymerase chain reaction for Shiga-like toxin-producing E. coli is presently being developed. Other diagnostic tests available are pathogenicity assays; ELISAs or DNA probes for detection of EPEC, EIEC, ETEC, and EHEC (including E. coli 0157:H7); and antimicrobial susceptibility testing, plasmid profiles, and other subtyping methods to determine the relatedness of enteric bacteria.

1. Stool. A pathogenic bacterium can be identified by isolating the

organism in culture, by serotyping, or by identifying a characteristic marker for virulence. Fresh stool specimens should always be used when possible to ensure that fastidious organisms and toxins that decompose easily are detected before they degenerate. Expedient refrigeration of specimens is important since bacteria can easily be overgrown by competing organisms in stool specimens left at room temperature for greater than 4 hours. If testing must be delayed beyond 48 hours after the specimen is collected, the specimen should be frozen to retard the overgrowth of bacteria. Since most bacterial pathogens can be cultured from appropriately acquired rectal swab specimens, rectal swabs or swabs of fresh stools are preferred to bulk stool specimens. This method of collection also facilitates storage and transport.

b. Serum. Specific antibody testing may be possible for some bacterial enteric agents such as Shigella. As is true for viral infections, antibody titers to bacterial agents generally rise by the end of the first week after onset of illness and peak by 3-4 weeks. A fourfold rise in specific antibody titer is accepted as diagnostic of a recent infection. C. Parasites

1. General guidelines Some tests available for formalin-preserved specimens

include the formalin-ethyl acetate concentration, which is used for both helminths and protozoa, and the kinyoun carbol-fuchsin(modified acid-fast)-stained slide, which is used for Cryptosporidium. PVA-preserved specimens are used to prepare Trichrome-stained slides and examined for protozoa.

1. Stool. Parasites are usually detected and identified with

microscopy of fresh or appropriately preserved stool specimen (although parasites can sometimes be grown on special media). Fresh specimens are needed for direct microscopy because trophozoites are fragile and may not survive environmental stress. The PVA and formalin fixatives preserve the morphology of cysts and trophozoites in stool specimens for diagnostic testing.

b. Serum. Unlike outbreaks of gastroenteritis due to bacteria or viruses, an outbreak of parasitic diarrheal illness can sometimes be identified with a convalescent-phase serum specimen only. However, it is sometimes necessary to have both acute and convalescent serum specimens to make a definitive diagnosis. V. CDC Contacts for Consultation

1. Epidemic Intelligence Service (EIS) Officer Viral Gastroenteritis Unit Respiratory and Enteric Virus Branch Division of Viral and Rickettsial Diseases Center for Infectious Diseases Centers for Disease Control Mailstop G-04, SB47, Bldg. 7 Atlanta, GA 30333 (404) 639-3040

2. EIS Officer Enteric Diseases Branch Division of Bacterial Diseases Center for Infectious Diseases Centers for Disease Control Mailstop C-09 Bldg. 1, Rm. 5428 Atlanta, GA 30333 (404) 639-3753

3. EIS Officer Epidemiology Activity Parasitic Diseases Branch Division of Parasitic Diseases Center for Infectious Diseases Centers for Disease Control Mailstop F-13 Bldg. 23, Chamblee Atlanta, GA 30333 (404) 488-4178

SELECTED BIBLIOGRAPHY Balows A, Hausler Jr. WJ, Lennette EH, eds. Laboratory diagnosis of infectious diseases: principles and practice. 1st ed. New York: Springer-Verlag, Inc., 1988. Benenson AS, ed. Control of communicable diseases in man. 14th ed. Washington, DC: American Public Health Association, 1985. Ciba Foundation. Novel diarrhea viruses. In: Ciba foundation symposium. New York: John Wiley & Sons, Inc., 1987:128. Cohen ML. Epidemiology of diarrheal disease: infectious diarrhea. Infect Dis Clin North Am 1988; 2(3): 557-70. Feigin RD, Cherry JD, eds. Gastrointestinal infections: textbook of pediatric infectious diseases. 2nd ed. Philadelphia: WB Saunders Co., 1987. Guerrant, RL. Gastrointestinal infections and food poisoning. In: Mandel GL, Douglas Jr RG, Bennet JE, eds. Principles and practice of infectious diseases. 2nd ed. New York: John Wiley & Sons, Inc., 1985:635-46. Guerrant RL, Hughes JM. Nausea, vomiting, and non-inflammatory diarrhea. In: Mandell GL, Douglas Jr. RG, Bennet JE, eds. Principles and practice of infectious diseases. 2nd ed. New York: John Wiley & Sons, Inc., 1985:646-55. Hughes JM. Food poisoning. In: Mandell GL, Douglas Jr. RG, Bennet JE, eds. Principles and practice of infectious diseases. 2nd ed. New York: John Wiley & Sons, Inc., 1985:680-91. Kapikian AZ, Chanock RM. Viral gastroenteritis. In: Evans AS, ed. Viral infections of humans: epidemiology and control. 3rd ed. New York: Plenum Medical Book Co., 1989. Manson-Bahr PEC, Bell DR. Diarrhea caused by protozoa. In: Manson's tropical diseases. 19th ed. London: Bailliere Tindall, 1987. Penaranda ME, Cubitt WD, Sinarachatanant P, Taylor DN, Linkanonsakul S, Saif L, Glass RI. Group C rotavirus infections in patients with diarrhea in Thailand, Nepal, and England. J Infect Dis 1989;160(3):392-7. Pickering LK, Cleary TG. Approach to patients with gastrointestinal infections and food poisoning. In: Feigin RD, Cherry JD, eds. Textbook of pediatric infectious diseases. 2nd ed. Philadelphia: WB Saunders Co., 1987:622-51. Tanowitz HB, Weiss LM, Wittner M. Diagnosis and treatment of protozoan diarrheas. Am J Gastroenterol. 1988;82(4):339-50. Thorne GM. Diagnosis of infectious diarrheal diseases: infectious diarrhea. Infect Dis Clin North Am 1988; 2(3): 747-74.

*Testing at CDC is for research purposes and/or outbreak evaluations in collaboration with state and local health departments. Specimens should not be sent to CDC without prior discussion a) first, with the appropriate state health department and b) second, with the appropriate CDC laboratory. If an outbreak of gastroenteritis occurs and CDC's assistance is needed, early contact with appropriate persons at CDC for information about specimen collection is strongly encouraged. *Preservatives are available in commercial kits. *Testing done at CDC is for research and/or outbreak situations. Contact the appropriate CDC staff member(s) to discuss any questions concerning outbreaks of gastroenteritis or methods of collecting specimens.

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