1. Laboratory Diagnosis of Infection
Satisfying Koch’s Second Postulate

2. Lesson objectives
Discuss general procedures for the collection and preservation of bacterial specimens for the diagnosis of an infectious disease.
Discuss the variables that can affect the quality of a specimen.
Relate bacterial growth requirements and generation time to specimen collection and processing.
Discuss specific specimen collection procedures to assist in the diagnosis of an infectious disease: wound, ear, eye, blood, urine, feces, and sputum.
Discuss the importance of the Gram stain and other staining procedures as a tool in determining specimen quality, identification, and empiric therapy.
Briefly discuss laboratory procedures used to isolate and identify infectious microorganisms.
Explain non-culture based methods for diagnosing infectious disease.

3. Diagnosis of infectious disease starts at the patient
Specimen collection: The following safeguards must always be in place when a specimen is collected for analysis in the microbiology lab: 1) asepsis:

4. Correct sample: any part of the body can be sampled for microbiological analysis. Each body part has specific considerations.
blood: collect 2 samples in special tubes or bottles from one venipuncture. Collect 2 more 30 minutes later, preferably from a different venipuncture site. Collect before antibiotic therapy. One bottle is aerobe, the other is anaerobe. If blood volume is limited, submit aerobe only. Disinfect skin with iodine or chlorhexidine (not alcohol).

5. Urine: may be collected through urinary catheter or “clean catch mid-stream” (ccms). Ccms: disinfect genital area. Collect specimen (at least 10 ml) midstream. Why not collect immediate void?
suprapubic catheter: catheter is surgically inserted through the top of the bladder. Advantages: a) bypasses the urethra b) can collect anaerobic urine specimen

6. Sputum: mucus obtained from lower respiratory system
Sputum: mucus obtained from lower respiratory system. Not generally produced by healthy individuals. May be produced during infection, cancer, trauma, or by smokers. Sputum is usually collected when the patient awakes (more concentrated). Methods of collection: a) deep coughing (with/without chest percussion) b) using a suction catheter (Luken’s Trap) can be used to collect anaerobic specimen.

7. Sputum must be distinguished from saliva
Sputum must be distinguished from saliva. Saliva contains a large variety of bacteria and fungi (“normal oropharyngeal flora”). If a sputum specimen contains abundant saliva, the microbes that grow will be those found in the mouth, and pathogens from the lungs may be missed. How to distinguish sputum from saliva: the Gram stain: saliva: many squamous epithelial cells, very few polymorphonucleocytes (neutrophils) or PMNLs, large variety and numbers of microbes.

8. This specimen should not be cultured. Lab will request re-collect
This specimen should not be cultured. Lab will request re-collect. Report “oropharyngeal contamination”
A good sputum sample will have large numbers of PMNLs and one or two bacterial types:
What to do? Many PMNLs and many epithelial cells? No PMNLs nor epithelial cells?

9. 3) Throat swab, wound culture: Use a moistened transport swab
3) Throat swab, wound culture: Use a moistened transport swab. The medium is a minimal medium—all needed nutrients, but 1/10 concentration. Keeps pathogens alive without promoting growth. Take care to culture only the site ordered. Throat swabs must not touch

10. Body fluids:
Synovial fluid (joints—viscous)
Peritoneal, pericardial, pleural
Exudate (unexpected fluid, such as in a cyst, abscess)
Cerebrospinal (csf) strict antisepsis of puncture area and aseptic technique is followed. Collected through needle aspiration, often by M.D. with nurse assisting. Note appearance (bloody, milky, cloudy, colored) Fluids should be considered “STAT” specimens.

11. Stool: Main concern with stool specimens is that the specimen does not contaminate the environment. Over 90 % of the stool’s mass is bacteria. Note the appearance of the stool (formed, loose, liquid). Also note any abnormalities (unusual color, presence of frank blood, visible parasites or undigested objects).
Reproductive specimens: (vaginal swab, discharge) Organisms that cause STIs are often fragile, and must be transported to lab quickly.

12. Specimens that cannot be transported to the lab immediately (within 15 minutes) should be refrigerated if delay is unavoidable. Exceptions:
Csf (meningitis organisms are killed by refrigeration)
Vaginal/penile specimens and discharges (agents of STI are killed by refrigeration)
Anaerobe specimens (refrigeration encourages oxygenation.)

13. Getting the specimen ready for the lab
Lab slip: sample on page accession number: corresponds to that specimen only. Other specimens from the same patient will have different accession numbers. - patient name and ID number (always confirm identity of patient by hospital bracelet)
Exact tests ordered—check specimen type
Date and time of specimen collection
Tentative diagnosis (indicate if diagnosis is confirmed or tentative. Patient diagnosis may determine how testing is done)
Medications (especially antibiotics)
STAT if ordered. Where/how should results be reported?

14. Now the lab has the specimen. Now what???
1) Direct examination of the specimen: Gram stain: early indicator that microbes are present and what they might be. Acid fast stain (AFB) (p. 55): Similar to Gram stain, but decolorizer is acidified alcohol. Detects the presence of Mycobacterium tuberculosis. India ink stain: Used on csf to detect budding yeast with capsules (Cryptococcus neoformans). Physical assessment of specimen (stool characteristics, appearance of urine).

15. Culture: Different specimens have different procedures for routine culture and sensitivity.
In general: prepare stains first culture onto selective and differential media. If using one swab, go from least selective to most selective. Inoculate broth last (if using a swab, break swab off into broth).

16. Reading culture results
Initial reading of plates: Pathogen identity can be narrowed down on the following basis: compare culture results to Gram stain: if one bacterial type (Gram positive cocci in chains) is noted, and only one colony type is seen, there’s a good chance they are the same organism. Specimen type: Certain pathogens are commonly found in certain parts of the body urine: E. coli, Proteus species csf: Neisseria meningiditis, Streptococcus pneumoniae sputum: Klebsiella pneumoniae, Hemophilis influenzae Biochemical tests: Organisms are further identified through a series of biochemical tests. Sometimes (though not often) these tests are related to the organism’s pathogenic characteristics.

17. Common biochemical tests: Oxidase: used to categorize Gram negative organisms Fermentation: Many Gram negative rods ferment sugars (use sugars without oxygen, producing acid). Which sugars are fermented give an indication to the identity of the microbe. In particular lactose fermentation separates many pathogenic Gram negative rods. Enzyme production: some bacteria are identified by the enzymes they excrete. Some of these are exotoxins, but not all: coagulase: Staphylococcus aureus is positive (pathogen), Staphylococcus epidermidis (non-pathogen) is negative. Some bacteria excrete enzymes that can be identified even without culture in rapid ID kits. C. dificile is diagnosed by exposing suspected stool to human cells in culture. The organism does not need to be grown.

18. What if the pathogen can’t be cultured?
Immunologic typing: If the pathogen can’t be grown outside the host, it is possible to detect antibodies to the pathogen using direct or indirect methods. Direct: latex agglutination for meningitis: csf is boiled and concentrated. It is then exposed to suspension of latex particles with antibodies to various pathogens. Positive result = agglutination. Negative result = no agglutination. Direct immunologic test detects antigens of the pathogen.

19. Indirect: ELISA for antibodies p. 423
Commercially prepared antigens are adsorbed to test plate. Wash excess
Patient’s serum sample is applied. Wash excess.
Antibodies to human IgG is applied (raised in a different animal—commercially prepared) This antibody is chemically bound to an enzyme.
Substrate for the enzyme is added. The enzyme/substrate complex is colored. A colored result is positive, no color is negative.

20. Genetic tests: It is possible to purchase DNA samples of a variety of pathogens. Only certain DNA sequences are available (therefore, the DNA is non-pathogenic). PCR: Polymerase Chain Reaction: If the pathogen is only present in small numbers (e.g. tuberculosis), it is possible to selectively clone large numbers of copies of the pathogen’s DNA. PCR is also used to identify agents of bioterrorism (anthrax) and to identify DNA samples in forensic analysis.

21. Lab Considerations
Quality Control: Every procedure, media, and piece of equipment in the laboratory must be routinely tested for its abilities to detect positive results and to determine negative results as negative. Durable equipment (incubators, refrigerators, autoclaves): Monitored daily for temperature, air quality, and asepsis. Records are maintained and audited.

22. Media: All growth media must be identified by lot number, and tested for sterility and performance. Reference organisms are maintained, cultured on new batches of media. Example: EMB agar: new batch must include culture of S. aureus (Gram positive), E. coli (Gram negative, lactose fermenter), P. vulgaris (Gram negative, non-lactose fermenter). Acceptable Quality: S. aureus does not grow
E. coli grows, green colonies
P. vulgaris grows, pink colonies

23. Test anomalies
False negative: Pathogen is present, test fails to detect it. If a false negative occurs, the test has low sensitivity.
False positive: Pathogen is not present, but test identifies it as present. If a false positive occurs, the test has low specificity.
Remedy: