How do you diagnose infectious disease?

Question 1

What are the 5 phases of clinical reasoning? and the 2 steps?

Answer 1

Step 1: Making a Diagnosis

1. History and physical examination
2. Diagnostic tests
   Microbiologic (this course)
   Others
3. Integration of clinical findings with test results

Step 2: Taking Appropriate Action

4. Weigh risks & benefits of alternatives
5. Consider patient preferences, develop treatment plan

Question 2

What do you need to know generally about patient history in suspected infection?

Answer 2

General:
Details about illness
- Symptoms
- Duration

Medications

• For chronic conditions
• For this illness

Allergies (especially antibiotics)

Family history

Question 3

What do you need to know specifically about patient history in suspected infection?

Answer 3

Specific
- Fever
TMAX and pattern, night sweats, rigors
- Infectious contacts
- Foreign travel
 Immunizations, prophylaxis, illness during/after
- Occupation
- Animal contacts
- Dietary history

Question 4

What are the non-micro investigations?

Answer 4

• Blood tests
  Blood counts
  Liver enzymes
  Coagulation studies

- Imaging
Plain x-rays
Ultrasound (including echocardiography)
CT, MRI
Nuclear medicine studies

Question 5

How do you come about the clinical diagnosis of infection? (3 things)

Answer 5

history + physical exam + diagnostic tests (blood, micro, imaging) = diagnosis

Question 6

What are the 4 (with one additional sub one) types of microorganisms?

Answer 6

bacteria
- mycobacteria
parasites
fungi
viruses

Question 7

What are current methods of detection for microorganisms?

Answer 7

Culture methods
Bacteria
- Mycobacteria

Fungi
- Yeasts
- Moulds = filamentous fungi
(Viruses)

Non-culture methods
Parasites
- Protozoa
- Helminths

Viruses
(Bacteria)
- (Mycobacteria)
(Fungi)

You need to have cells that viruses can take over to perpetuate for virus culture and that is difficult.

Question 8

What are tools for micro. lab diagnosis?

Answer 8

Microscopy

Culture

Serodiagnosis

• Antibody detection
• Antigen detection

Molecular methods

• Amplification = PCR, others
• Nucleic acid probes

Question 9

For lab diagnosis of bacterial infections, how is this done for gram stain + microscopy?

Answer 9

1. Laboratory Diagnosis of Bacterial Infections

 Methods vary depending on specimen type / suspected bacteria

1. Gram stain + microscopy
   - Fast turnaround, but not very sensitive (need lots of bacteria)
   - Good for specimens that should be sterile in healthy people
   - not very useful if lots of normal flora present
   - only tells you that bacteria are present – doesn’t ID the species

Fecal material is one where you would expect very large numbers of bacteria. Urine will have very very little numbers and they would be difficult to hear.

Question 10

For bacterial infections what comments are there on doing a culture?

Answer 10

2. Methods requiring growth (culture)
   
   • Place specimen on appropriate culture media to allow growth of any bacteria that may be present
   • Additional work-up performed on significant organisms:
     
     • Identify species
     • using phenotypic characteristics (metabolic end products, growth properties, production of enzymes, etc.)
     • using protein profiles (mass spectroscopy)
       
       • Susceptibility testing (Susceptibility testing – what can they use to kill the bacteria to make the patient better.
         )

   Slow (usually 24-72 hrs.; but up to weeks for slow-growers )

   Only works if bacteria can be grown on laboratory media

   Contamination with normal flora makes things more difficult

   Still the most often used diagnostic method in micro labs

Question 11

What are methods not requiring growth for bacterial infections?

Answer 11

3. Methods not requiring growth
    Nucleic acid based tests
   
   • Detect a DNA sequence unique to the bacteria
     (eg. PCR - polymerase chain reaction)
      fast, very sensitive, but technically complex & expensive

 Serological tests (ELISA assays)
- Look for antibodies in blood produced against a bacteria
 Does not distinguish between current vs. past infection
- Look for bacterial antigens using prepared antibodies

 Note:  Non-growth methods require that you know exactly what 	  	       bacterial species to look for  (not a “screening” test)

Question 12

What are the problems with lab diagnosis of viral infections and what are 3 methods for doing this?

Answer 12

Problems:

  Symptoms of some viral diseases mimic other microbial diseases

  Different types of viruses may produce very similar symptoms

  Culture methods are difficult because viruses only “grow” inside living cells; increasingly are replaced by molecular assays

Methods:

1. Serology - detects antibodies in blood produced against a virus
    some assays (often IgM) indicate current or recent infection
    others (often IgG) useful as a screen to determine if someone has been exposed in the past
    Acute and convalescent: >4-fold IgG rise could indicate recent infx’n
2. Molecular (PCR) - detects viral RNA / DNA in patient specimens
    rapid, highly sensitive and specific
    complex, expensive, and not good for large #’s of specimens
   • Advancing technology may remove these barriers

IgM is the earliest antibody formed, so if this is present that would indicate acute infection. There are some cases where the IgM can stay around for even a year.
IgG can be used to indicate possible reactivation in the future like chicken pox, if you have the IgG for this then it is possible that you could develop shingles.

3. (Direct or Indirect) Fluorescent Antibody (DFA or IFA) tests
   - Looks for virus-infected cells in a patientDFA:  Collect patient specimen likely to contain infected cells

              Add antibodies (tagged with a fluorescent dye) that recognize 	         	       viral surface components, then wash
   
              Examine microscopically for binding of fluorescent Ab to cell   Fast, but not very sensitive (need lots of infected cells)
        -  Requires skilled microscopist

Question 13

What are approaches to lab diagnosis of fungal infections?

Answer 13

• Direct microscopic examination
   Examine tissue or other specimen and detect the presence of fungal elements = usually yeasts or hyphae

   -  Culture (moulds) 
         Culture media and temperature that support optimal growth, 	       	  macroscopic and microscopic appearance
   - Biochemical identification (yeasts)
      Similar to bacteria
   - Non-culture methods (yeasts and moulds)
     PCR-sequencing, serology (dimorphics), 
   antigen detection (histo, crypto), mass spectroscopy

Question 14

What are some laboratory diagnosis of parasitic infections?

Answer 14

 Almost exclusively by microscopic examination

        - requires appropriate specimen likely to contain parasite
        - requires knowledge of parasite morphology
        - time consuming and technically demanding	

	   Stool EIA available for detection of Giardia lamblia and 		    	     Cryptosporidium species from stool

	  Serologic assays available to detect exposure to certain 	  	    	    parasites

Question 15

What kind of turnaround can you expect for different tests?

Answer 15

What kind of turn-around time can you reasonably expect?

  Gram stain  -  1 to 2 hrs

  Bacterial culture with -  24 to 48 hrs
	- susceptibility  48 to 72h

  Mycobacterial culture with susceptibility  -  2 to 8 weeks 

  Viral serology or DFA  -  4 to 24 hrs

  Mycology (fungi)  -  24 hrs to 6 weeks

  Parasites  -  24 hrs to 1 week

  Any molecular (PCR) test  -  4 to 24 hrs

Factors beyond the lab’s control 

             	- Transportation delays
	- Correct specimen?;  Properly labeled, etc.? 
	- If you want it “STAT”, does the requisition say so?
		- BUT if everything is STAT, then nothing is STAT!

Collect the right specimen at the right time in the right way

Question 16

What is the right specimen and what does it mean to be at the right time?

Answer 16

The Right Specimen:

 One that has a reasonable expectation of containing the pathogen

-   Mainly dependent on symptoms 
         eg.  Gastrointestinal  =  feces
	      Respiratory  =  sputum, nasopharyngeal swab or aspirate
	      Neurological  =  cerebrospinal fluid

         - Sometimes dependent on testing methodology
         eg. Serological methods require blood specimen

The Right Time:

 Preferably before administration of antibiotics, antivirals, etc.

  	- May interfere with growth-based testing methods
- But do not delay treatment for life-threatening infections (e.g. 	  	bacterial meningitis) while arranging for tests 

 Collect specimen while patient is still symptomatic
- Microbe may no longer be present very late in disease

Question 17

What is the right way to get a specimen?

Answer 17

The Right Way:

 Use the proper specimen collection device

       - Swabs +/- “transport media”                                                   	 	 	    (ensures viability during transport to lab)
       - Proper containers for urine, feces, etc.  

 Remember that all such devices are sterile before use
- Avoid introducing your own normal flora

 Use proper collection technique

       - Representative of area to be sampled
       - Get an adequate volume (pus is better than a swab)

 If self-collected, ensure that patient is properly educated
eg. “mid-stream” vs. “first catch” urine

 Label specimen properly, complete the requisition, transport to lab

                         If unsure, call the laboratory!!