Laboratory Diagnosis: An Overview

1. Laboratory Diagnosis: An Overview

2. Goals • Provide an overview of pathogens tested in public health laboratories • Describe laboratory tests commonly used in outbreak investigations

3. A Review of Specimens • Laboratory staff analyze specimen to determine presence or absence of suspected pathogens • Specimens can tell us: • Whether different individuals are infected with the same pathogen • Whether a particular source is causing an outbreak

4. A Review of Specimens • Environmental samples include: • food (items suspected in a foodborne outbreak) • water (from a lake, water supply, or drinking fountain) • surfaces (medical equipment, countertops, etc.)

5. A Review of Specimens • Proper specimen collection is important (see FOCUS Volume 4, Issue 2) • Right sample must be collected • Collected in proper medium for survival • Transported within proper time frame and temperature • Accompanied by appropriate information

6. Microorganisms • Bacteria: single-celled organisms • Examples: Salmonella, Streptococcus (“strep”), Staphylococcus (“staph”), Escherichia Coli (E. coli) • Viruses: DNA (or RNA) surrounded by protective coat of proteins • Examples: Influenza, HIV, West Nile, Noroviruses, common cold viruses (Coronavirus, Rhinovirus) • Other pathogens: toxins produced by bacteria, parasites, fungi, chemicals

7. Why is Lab Diagnosis Necessary? • Lab identification of the agent is crucial: • Because diagnosis should not be based on clinical symptoms alone • Many agents cause similar symptoms • Clinical symptoms may be unclear or too general • Physicians might not recognize a rare disease • To connect individual cases in outbreak • To ensure proper medical treatment for patients • Norovirus and Shigella infections cause same symptoms: Norovirus treatment is symptomatic relief; Shigella can be treated with antibiotic

8. Why is Lab Diagnosis Necessary? • Sometimes necessary to conduct further studies to determine specific strain or serotype: a.k.a. subtyping • Dozens of strains of Noroviruses (e.g., including Hawaii virus, Snow Mountain virus, Desert Shield virus, Toronto virus); if people infected with a Norovirus have different strains, the infections are unrelated

9. Why is Lab Diagnosis Necessary? • To help identify outbreaks across state lines • 2006: CDC officials notified of several small clusters of E. coli 0157:H7 infections in Wisconsin and Oregon, with fresh spinach implicated as the probable source. The same day, New Mexico epidemiologists contacted Wisconsin and Oregon epidemiologists regarding similar cluster of infections. CDC’s PulseNet confirmed through laboratory testing that E. coli O157:H7 strains from infected patients in Wisconsin had same PFGE pattern and identified that pattern in patients from several other states.

10. Laboratory Diagnosis and Surveillance Programs • Council of State and Territorial Epidemiologists and CDC recommend surveillance for list of pathogens • Each state decides which pathogens healthcare providers and laboratories must report • Lab reports to the state health department using disease reporting system • Guidelines specify which identification methods are used to ensure that only confirmed cases are reported • State lab responsible for identification when local labs do not have necessary expertise • State lab has final responsibility for reporting cases to state health department • If identification not possible at the state level, CDC may be asked to help

11. Pathogen Identification and Typing • Method depends on the type of organism • Some methods are well established for particular organisms • Guidelines exist for identifying the organism

12. Pathogen Identification and Typing

13. Pathogen Identification and Typing

14. Microscopy • Useful for larger organisms such as bacteria or fungi • For standard optical or light microscope • Small part of specimen smeared onto glass slide • Stains applied to help identify cells and substances within the specimen • When using Gram stain, “Gram-positive” bacteria have a cell wall that will stain purple while “Gram-negative” bacteria stain as red

15. Microscopy • Common bacteria shapes: • Round (cocci) • Rod-shaped (bacilli) • Bacteria can cluster in pairs, chains, other arrangements • E. coli is a Gram-negative rod • S. pneumoniae or pneumococcus is a Gram-positive diplococcus, a round bacterium that clusters in pairs • Shapes and growth patterns also used to identify fungi and fungal spores

16. Microscopy • Viruses are much smaller than bacteria or fungi, require a very high degree of magnification • Electron microscope shoots electrons at virus (like a camera flash shoots light at an object to capture the image) • Many viruses have a characteristic shape, can be identified from microscope image

17. Culture • Provide right temperature, moisture, and nutrients for a pathogen to thrive and replicate, introduce a sample, wait for growth • Case definition may require a definite case to be “culture confirmed” • Outbreak of E. coli O157:H7 infections among Colorado residents in June 2002, part of the case definition was that specimens taken from patients were culture-positive for E. coli. Contaminated beef was implicated and over 350,000 pounds of beef were recalled • Can increase amount of organism to perform other types of tests

18. Culture • Bacteria often grown on a Petri dish • Plate containing growth medium (gelatin-like substance called agar, nutrients other materials) • Bacteria form distinctive-looking colonies Culture of Nocardia asteroids, a mycobacterium commonly found in soils. It causes illness in people with defects in cellular immunity.

19. Culture • Some bacteria grow inside the culture nutrients: stab culture • Test tube filled with agar and nutrients, sterile wire is dipped into sample and stabbed into tube Stab culture of Legionella pnuemophila, the agent that causes Legionnaire’s disease. It is found in aqueous environments.

20. Culture • Viruses need living cells to reproduce, so often grown in tissue culture derived from growing cells or tissues. • May be tested by nucleic acid-based methods or viewed under an electron microscope • June 2003: Multistate monkeypox outbreak, with monkeypox virus isolated from multiple patients and cultured. All case patients found to have links to prairie dogs. Virus from patients grown in cell culture and confirmed using electron microscopy.

21. Culture • Different organisms require different conditions • Not all organisms can be grown in culture; other methods must be used • Requires considerable amount of time to grow certain organisms, can slow investigation • Pulmonary blastomycosis (fungal infection that causes severe respiratory symptoms) can require up to 5 weeks in culture before confirmatory diagnostic tests can be performed

22. Culturing a Clinical Specimen • A clinical specimen is cultured for microorganisms known to thrive in the particular environment and associated with certain clinical symptoms • Fecal samples in diarrheal illnesses are cultured for enteric pathogenic bacteria, including Salmonella serotypes (typhi, enteritidis, typhimurium, etc.), Shigella, Campylobacter, Yersinia, Escherichia coli 0157:H7, Vibrio • Respiratory samples are cultured for pathogens such as Streptococcus pneumoniae, Bordetella pertussis, Haemophilus influenzae, Influenza, Legionella, mycobacterium • Cervical, vaginal or penile specimens may be cultured for Neisseria gonorrhoeae, herpes, other organisms that cause genital infections

23. Serology • Uses immune response to determine whether a person has fought off an infection by a particular pathogen • Compare blood samples taken at the time of exposure (or shortly thereafter) and weeks later • Looks at antibodies, or immunoglobulins • If no antibodies are present (or present in early form) at first blood sample and fully mature antibodies are present at second sample, person has been recently exposed • Example: syphillis rapid plasma reagin (RPR) test detects presence of antibodies against syphilis in a blood sample

24. Serology • Limitations: • Not useful for a rapid intervention • Often difficult to obtain a blood sample even once, let alone twice • May be useful: • When pathogen is not easily detected in other types of samples • When source of exposure has been eliminated with no remaining sample to test • For research purposes 

25. Antigen Detection • Small parts of a viral or bacterial pathogen • Separate antigens from other material, use antibodies to find a particular antigen • If antibodies attach to the target antigen, pathogen has been identified • If the antibodies do not find anything to attach to, do not know which organism is causing infection • Many ways antigens can be separated from other matter in a specimen, many ways antigen test can be performed

26. Phage Typing • Short for bacteriophage, a virus that infects bacteria • Each type of phage attacks a particular type of bacteria • Most often used to identify strains of Staphylococcus aureus • A “known” phage mixed with “unknown” bacterium, poured onto an agar plate, allowed to grow • If bacteria are correct strain, a plaque will form • If no plaques, bacteria can be eliminated as possible pathogen A “gamma phage” is used to identify Bacillus anthracis growing on agar plate. Lawn of bacteria interrupted where the gamma phage has attacked the bacteria, causing a “plaque,” or hole in the bacterial growth.

27. Molecular techniques • Every pathogen has DNA, RNA, or both • Can test a sample for presence of a bacteria or virus by looking for the DNA • Often referred to as “molecular methods” • Useful for distinguishing between strains • Can distinguish between strains of E. coli normally found in the human gut and a pathogenic strain causing disease • Identifying exact strain is important for finding source of an outbreak

28. Summary • This overview of diagnostic techniques can give you a better sense of what happens once you send that specimen off to the laboratory • Future issues of FOCUS will delve further into more advanced laboratory techniques, such as molecular identification and typing

29. Additional Resources • To see examples of microorganisms that can often be identified with a Gram stain, go to http://www.uphs.upenn.edu/bugdrug/ antibiotic_manual/gram.htm and click on “Typical Gram stains.” • To see electron micrographs of viruses, go to http://www.ncbi.nlm.nih.gov/ICTVdb/Images/index.htm. • To find information on the diseases most often tested at public health labs, visit the North Carolina State Laboratory of Public Health Microbiology Web site: http://204.211.171.13/Microbiology/default.asp. • To find infectious disease information from the National Center for Infectious Diseases, go to http://www.cdc.gov/ncidod/diseases/index.htm. • To use the American Society for Microbiology Microbe Library, visit http://www.microbelibrary.org.

30. References • Centers for Disease Control and Prevention. Ongoing multistate outbreak of Escherichia coli serotype O157:H7 infections associated with consumption of fresh spinach --- United States, September 2006. MMWR Morb Mort Wkly Rep. 2006; 55(Dispatch):1-2. Available at: http://www.cdc.gov/mmwr/preview/mmwrhtml/ mm55d926a1.htm. Accessed December 8, 2006. • Centers for Disease Control and Prevention. Multistate outbreak of Escherichia coli O157:H7 infections associated with eating ground beef --- United States, June--July 2002. MMWR Morb Mort Wkly Rep. 2002;51:637—639. Available at: http://www.cdc.gov/mmwr/ preview/mmwrhtml/mm5129a1.htm. Accessed November 30, 2006. • Centers for Disease Control and Prevention. Multistate outbreak of monkeypox—-Illinois, Indiana, and Wisconsin, 2003. MMWR Morb Mort Wkly Rep. 2003;52:537-540. Available at: http://www.cdc.gov/ mmwr/PDF/wk/mm5223.pdf. Accessed November 30, 2006.

31. References • Martynowicz MA, Prakash, UBS. Pulmonary blastomycosis: An appraisal of diagnostic techniques. Chest. 2002;121:768-773. • Mayer G. Bacteriology Chapter 7: Bacteriophage. In: University of South Carolina School of Medicine. Microbiology and Immunology On-line [Internet]. September 11, 2003. Available at: http://www.med.sc.edu:85/mayer/phage.htm. Accessed November 30, 2006. • Herwaldt, et al. Microbial Molecular Techniques. In: Epidemiologic Methods for the Study of Infectious Diseases, JC Thomas, DJ Weber, eds. Oxford University Press, 2001: 163-191.