Difference between revisions of "Course:PATH4172017W2/Case 3"

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(Question 3)
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(1) Jorgensen, J. H., & Pfaller, M. A. (Eds.). (2015). Manual of clinical microbiology. Retrieved from http://ebookcentral.proquest.com
 
(1) Jorgensen, J. H., & Pfaller, M. A. (Eds.). (2015). Manual of clinical microbiology. Retrieved from http://ebookcentral.proquest.com
  
(2) Garcia, Isenberg & Knovel, 2010  
+
(2) Garcia, L. S. & Isenberg, H. D. (2010). Clinical Microbiology Procedures Handbook, Volumes 1-3 (3rd Edition). American Society for Microbiology (ASM). Available from: https://app.knovel.com/hotlink/toc/id:kpCMPHVE03/clinical-microbiology/clinical-microbiology
  
 
(3) Bram M. W. Diederen, Jan A. J. W. Kluytmans, Christina M. Vandenbroucke-Grauls, Marcel F. Peeters. Utility of real-time PCR for diagnosis of legionnaires’ disease in routine clinical practice. Journal of Clinical Microbiology. 2008;46(2):671-677. http://jcm.asm.org/content/46/2/671.abstract. doi: 10.1128/JCM.01196-0
 
(3) Bram M. W. Diederen, Jan A. J. W. Kluytmans, Christina M. Vandenbroucke-Grauls, Marcel F. Peeters. Utility of real-time PCR for diagnosis of legionnaires’ disease in routine clinical practice. Journal of Clinical Microbiology. 2008;46(2):671-677. http://jcm.asm.org/content/46/2/671.abstract. doi: 10.1128/JCM.01196-0
Line 105: Line 105:
 
(7) PCR amplification. Promega Web site. https://www.promega.ca/resources/product-guides-and-selectors/protocols-and-applications-guide/pcr-amplification/. Updated 2018.  
 
(7) PCR amplification. Promega Web site. https://www.promega.ca/resources/product-guides-and-selectors/protocols-and-applications-guide/pcr-amplification/. Updated 2018.  
  
(8)       Nazarian, Bopp, Saylors, Limberger & Musser, 2008
+
(8) Nazarian, E. J., Bopp, D. J., Saylors, A., Limberger, R. J., & Musser, K. A. (2008). Design and implementation of a protocol for the detection of Legionella in clinical and environmental samples. Diagnostic microbiology and infectious disease, 62(2), 125-132
  
 
(9) Stellwagen NC. Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution. Electrophoresis. 2009;30(Suppl 1):S188-S195. doi:10.1002/elps.200900052.
 
(9) Stellwagen NC. Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution. Electrophoresis. 2009;30(Suppl 1):S188-S195. doi:10.1002/elps.200900052.

Revision as of 19:22, 8 March 2018

PATH 417
Case Projects
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Instructors
Niamh Kelly
Case Projects
Questions?

Discussion for This Page

Case 3

A Cruise Holiday

To celebrate Tom’s retirement his wife and two adult children accompany him on a long anticipated cruise. Tom’s asthma flares up a few days before the cruise but with a corticosteroid nebulizer in tow he feels well enough to join the cruise. Even more than the rest of his family, Tom enjoys the various hot tubs aboard the massive ship those first few days, relishing the relaxation after a busy final year at work.

On the fifth day of the cruise, Tom wakes up in a sweat with a cough that continues throughout the day. As the day wears on he feels worse with a headache, muscle aches and nausea accompanying the cough. His wife arranges for the cruise doctor to visit him in his cabin. The doctor examines Tom, notes his high temperature, relatively nonproductive cough and recent history of asthma and corticosteroid therapy. She takes a full history including taking note of his activities during the first days of the cruise and diagnoses Tom with a pneumonia. She explains that her presumptive diagnosis is that of Legionnaires disease and leaves Tom’s wife with a sterile sample container to collect whatever fluid Tom might cough up for delivery to her. She explains that she can do a microscopic examination on the respiratory fluid which will help in the diagnosis.

In the meantime she starts Tom on erythromycin and lets the family know that she will check in on Tom regularly over the next few days to monitor his progress. More people are diagnosed with a similar pneumonia over the next two days, mostly in people who came aboard with a slightly compromised immune system, like in Tom’s case. The cruise ship alerts the hospital at their next port of call in case any of the patients worsen enough to require hospitalization. When they arrive at port blood samples are collected from all of the patients and delivered to the hospital laboratory for serology. The ship also takes extra time in port to allow for a full scale sterilization regime to be performed on all of the hot tubs. At this stage Tom is feeling well enough to continue on the cruise, although at a slower pace than when he first boarded.

The Body System Questions

  1. What are the signs (objective characteristics usually noted/detected by a healthcare professional) and symptoms (subjective characteristics experienced by the patient).
  2. Which body system is effected. In what way has the normal physiological functioning of this area of the body been disturbed by the infection (without going into detail on the cause of this disturbance as this will be dealt with in questions 3 and/or 4). Representing this diagrammatically is always helpful. What made Tom susceptible to this infection?
  3. Why did the doctor prescribe erythromycin and how does this antibiotic work to rid the body of the organism?
  4. Would this be considered an ‘outbreak’, is it reportable and to what official body. What role might the hot tub have played in this infectious scenario?

The Microbiology Laboratory Questions

  1. Other than the stated bacterial cause, what are the most common bacterial pathogens associated with this type of infectious scenario.
  2. What are all the samples that could be taken for laboratory testing (including the blood and ‘sputum’ in this case) and how important is the Microbiology Laboratory in the diagnosis of this particular infectious disease?
  3. Explain the tests that will be performed on the samples in order to detect any of the potential bacterial pathogens causing this disease.
  4. What are the results expected from these tests allowing for the identification of the bacteria named in this case.

Bacterial Pathogenesis Questions

Using the following pathogenic steps outline the pathogenesis of the bacteria named as being responsible for this infection.

  1. Encounter: where does the organism normally reside, geographically and host wise, and what are the bacterial characteristics that leave it suited to these places of residence. How would our patient have come in contact with this bacteria
  2. Entry: what facilitates the entry of the bacteria into the human host? What are the molecular, cellular and/or physiological factors at play in the initial entry/adherence step from the point of view of the organism and the host.
  3. Multiplication and Spread: does the organism remain extracellular or do they enter into cells and what are the molecular and cellular determinants of these events. Do the bacteria remain at the entry site or do they spread beyond the initial site i.e. are there secondary sites of infection and why do the bacteria hone in on these particular secondary sites.
  4. Bacterial Damage: do the bacteria cause any direct damage to the host (or is the damage fully attributable to the host response, as indicated below) and, if so, what is the nature of the bacterial damage. Can it be linked to any of the signs and symptoms in this case

The Immune Response

  1. Host response: what elements of the innate and adaptive (humoral and cellular) immune response are involved in this infection.
  2. Host damage: what damage ensues to the host from the immune response?
  3. Bacterial evasion: how does the bacteria attempt to evade these host response elements.
  4. Outcome: is the bacteria completely removed, does the patient recover fully and is there immunity to future infections from this particular bacteria ?

Reports

A Cruise Holiday

To celebrate Tom’s retirement his wife and two adult children accompany him on a long anticipated cruise. Tom’s asthma flares up a few days before the cruise but with a corticosteroid nebulizer in tow he feels well enough to join the cruise. Even more than the rest of his family, Tom enjoys the various hot tubs aboard the massive ship those first few days, relishing the relaxation after a busy final year at work.

On the fifth day of the cruise, Tom wakes up in a sweat with a cough that continues throughout the day. As the day wears on he feels worse with a headache, muscle aches and nausea accompanying the cough. His wife arranges for the cruise doctor to visit him in his cabin. The doctor examines Tom, notes his high temperature, relatively nonproductive cough and recent history of asthma and corticosteroid therapy. She takes a full history including taking note of his activities during the first days of the cruise and diagnoses Tom with a pneumonia. She explains that her presumptive diagnosis is that of Legionnaires disease and leaves Tom’s wife with a sterile sample container to collect whatever fluid Tom might cough up for delivery to her. She explains that she can do a microscopic examination on the respiratory fluid which will help in the diagnosis.

In the meantime she starts Tom on erythromycin and lets the family know that she will check in on Tom regularly over the next few days to monitor his progress. More people are diagnosed with a similar pneumonia over the next two days, mostly in people who came aboard with a slightly compromised immune system, like in Tom’s case. The cruise ship alerts the hospital at their next port of call in case any of the patients worsen enough to require hospitalization. When they arrive at port blood samples are collected from all of the patients and delivered to the hospital laboratory for serology. The ship also takes extra time in port to allow for a full scale sterilization regime to be performed on all of the hot tubs. At this stage Tom is feeling well enough to continue on the cruise, although at a slower pace than when he first boarded.

The Body Systems Questions

Question 1

What are the signs (objective characteristics usually noted/detected by a healthcare professional) and symptoms (subjective characteristics experienced by the patient).

Question 2

Which body system is effected. In what way has the normal physiological functioning of this area of the body been disturbed by the infection (without going into detail on the cause of this disturbance as this will be dealt with in questions 3 and/or 4). Representing this diagrammatically is always helpful. What made Tom susceptible to this infection?

Question 3

Why did the doctor prescribe erythromycin and how does this antibiotic work to rid the body of the organism?

Question 4

Would this be considered an ‘outbreak’, is it reportable and to what official body. What role might the hot tub have played in this infectious scenario?


The Microbiology Laboratory

Question 1

Other than the stated bacterial cause, what are the most common bacterial pathogens associated with this type of infectious scenario.

Question 2

What are all the samples that could be taken for laboratory testing (including the blood and ‘sputum’ in this case) and how important is the Microbiology Laboratory in the diagnosis of this particular infectious disease?

Question 3

Explain the tests that will be performed on the samples in order to detect any of the potential bacterial pathogens causing this disease.

Culture

Cultures are considered the gold standard for Legionella diagnosis and are commonly performed to confirm the presence of L. pneumophila. Aspirates, sputum, bronchoalveolar lavage fluid and pleural fluid all have the potential to contain Legionella bacteria and are therefore all appropriate samples to perform a culture on. As the species of bacteria causing infection, if any at all, is unknown, a Gram stain should first be done to confirm the presence of any Gram negative, small and filamentous rods, characteristic indicators of Legionella infection. (1). During this process, the chosen media should be liquefied, then cooled solified before subsequently adding the sample; notably, MacConkey agar is used commonly for Gram negative bacterial isolation. During incubation, the plate should be kept at room temperature away from direct light. (2).

Once a Gram stain has been completed and the species is confirmed to be Gram negative, samples should be incubated in a cell culture plate in buffered charcoal yeast extract and α-ketoglutarate. Importantly, this medium provides essential nutrients for Legionella growth, including iron and L-cysteine. The bacteria may also be grown on trypic soy blood agar, or buffered charcoal yeast extract lacking L-cysteine, as a control; lack of growth would support the hypothesis of Legionella presence as L-cysteine is essential for growth. (1). Optionally, the microbiologist may add antimicrobial agents to select against normal flora that or other bacteria that may be residing in the sample. The plate should be incubated at 35 °C for optimal growth conditions. Notably, longer incubation times are needed as Legionella is a slower-growing bacterium. (3) Results are often obtained within 3-5 days of plating, rendering this diagnostic test less convenient than quicker tests, such as DFA staining and the Urinary Antigen Test. Sensitivity ranges from 10-80%, and so may produce more false negatives and false positives than other more reliable testing methods. (4)

A legionella culture is performed here on black charcoal yeast extract with alpha-ketoglutarate.

In order to increase probability of Legionella recovery, selective pressures against normal flora that may be present in the sample can be introduced. Cycloheximide, polymyxin B and vancomycin are all toxic additives that do not inhibit Legionella growth but may hinder growth of other bacteria. Acid and heat exposure may also be used as Legionella is more tolerant of these environmental factors than some normal flora. (1).

Once a culture has been completed, a confirmation of L. pneumophila presence can be completed using matrix-assisted laser desorption ionization – time of flight (MALDI-TOF), a form of mass spectrometry. PCR may also be used to confirm the presence of genes specific to L. pneumophila. (4)  

PCR, Gel Electrophoresis and Sequencing

Polymerase Chain Reaction, or PCR, is used as a tool to amplify areas of the Legionella genome. (5) Many samples can be used for PCR analysis, including sputum, bronchioalveolar lavage fluid, serum and urine, as these samples may contain whole or lysed bacterial contents that can be used for diagnosis. Notably, this technique is favourable in that it does not necessarily require a sputum sample, which not all patients produce during infection. In this PCR reaction, primers to known Legionella genes of interest are added to a sample, along with a DNA polymerase to amplify the region of interest. The sample is heated to 94°C to denature the DNA. Then, temperature is reduced to 40-60 °C, an ideal temperature for primers to anneal to the single-stranded DNA. Temperature is once again raised to 70-74°C to facilitate extension of the new strand via the function of DNA polymerase. This process is repeated continuously until the regions of DNA have been amplified extensively and are at detectable levels. Positive and negative controls are also used for comparison of the level of PCR product to the experimental samples. (6) Primers prepared for Legionella PCR will often be prepared for Legionella ribosomal RNA genes, including 16S rRNA, 5S rRNA, and 23S rRNA, and the macrophage infectivity potentiator gene, or mip. (7) Advantageously, as mip is relatively conserved between serogroups, it can be used to detect Legionella serogroups 1-16. (8)However, because these genes have been reported in studies as having specificity of lower than 99% in PCR reactions, it is not often considered a reliable independent diagnostic test, and may be coupled with other tests for confirmation. (6)

The results can be visualized using gel electrophoresis. PCR product is run through an agarose gel for 2-3 hours, and are then visualized under UV light in order to detect the amount of PCR product. Agarose gel is advantageous in that it is more porous than polyacrymide gel. As large amounts of DNA fragments are produced during PCR, this large pore size allows molecules to run quickly through the gel so that a size estimate of the genes can be reached.(9) Bands are produced on the gel based on size, with the smallest sized products moving further down the gel due to decreased resistance. Thus, the base pair length of the genes of interest must be known in order to confirm that the gene of interest has been amplified. (10)

Nucleotides are added to the primers after denaturing of DNA to allow an amplification of the strand of interest. This process is repeated until many copies of Legionella genes have been created.

To confirm the presence of Legionella DNA, the DNA is sequenced. Sanger sequencing methods are more likely to be used in this case than next generation sequencing as only one fragment is being amplified. It is then compared to Legionella genes located in databases such as NCBI BLAST. BLAST contains a database of known genes in strains of organisms, and allows one to infer homology based on the alignment and overlap of sequenced genes to genes in the database.

(1) Jorgensen, J. H., & Pfaller, M. A. (Eds.). (2015). Manual of clinical microbiology. Retrieved from http://ebookcentral.proquest.com

(2) Garcia, L. S. & Isenberg, H. D. (2010). Clinical Microbiology Procedures Handbook, Volumes 1-3 (3rd Edition). American Society for Microbiology (ASM). Available from: https://app.knovel.com/hotlink/toc/id:kpCMPHVE03/clinical-microbiology/clinical-microbiology

(3) Bram M. W. Diederen, Jan A. J. W. Kluytmans, Christina M. Vandenbroucke-Grauls, Marcel F. Peeters. Utility of real-time PCR for diagnosis of legionnaires’ disease in routine clinical practice. Journal of Clinical Microbiology. 2008;46(2):671-677. http://jcm.asm.org/content/46/2/671.abstract. doi: 10.1128/JCM.01196-0

(4) McGrath B, Drake J. Bronchoscopy in the ICU: An overview of broncho-alveolar lavage and bronchial washing  Airway eLearning.2014. http://www.airwayelearning.com/awel/articles/articles-1.aspx?Action=1&NewsId=2115&M=NewsV2&PID=71655.

(5) Chen DJ, Procop GW, Vogel S, Yen-Lieberman B, Richter SS. Utility of PCR, culture, and antigen detection methods for diagnosis of legionellosis. Journal of clinical microbiology. 2015;53(11):3474-3477. http://www.ncbi.nlm.nih.gov/pubmed/26292304. doi: 10.1128/JCM.01808-15.

(6) Den Boer J, Yzerman E. Diagnosis of legionella infection in legionnaires’ disease. Eur J Clin Microbiol Infect Dis. 2004;23(12):871-878. http://www.ncbi.nlm.nih.gov/pubmed/15599647. doi: 10.1007/s10096-004-1248-8.

(7) PCR amplification. Promega Web site. https://www.promega.ca/resources/product-guides-and-selectors/protocols-and-applications-guide/pcr-amplification/. Updated 2018.

(8) Nazarian, E. J., Bopp, D. J., Saylors, A., Limberger, R. J., & Musser, K. A. (2008). Design and implementation of a protocol for the detection of Legionella in clinical and environmental samples. Diagnostic microbiology and infectious disease, 62(2), 125-132

(9) Stellwagen NC. Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution. Electrophoresis. 2009;30(Suppl 1):S188-S195. doi:10.1002/elps.200900052.

(10) D S Lindsay, W H Abraham, R J Fallon. Detection of mip gene by PCR for diagnosis of legionnaires' disease. Journal of Clinical Microbiology. 1994;32(12):3068-3069. http://jcm.asm.org/content/32/12/3068.abstract.

Question 4

What are the results expected from these tests allowing for the identification of the bacteria named in this case.


Bacterial Pathogenesis Questions

Using the following pathogenic steps outline the pathogenesis of the bacteria named as being responsible for this infection.

Question 1

Encounter: where does the organism normally reside, geographically and host wise, and what are the bacterial characteristics that leave it suited to these places of residence. How would our patient have come in contact with this bacteria

Question 2

Entry: what facilitates the entry of the bacteria into the human host? What are the molecular, cellular and/or physiological factors at play in the initial entry/adherence step from the point of view of the organism and the host.

Question 3

Multiplication and Spread: does the organism remain extracellular or do they enter into cells and what are the molecular and cellular determinants of these events. Do the bacteria remain at the entry site or do they spread beyond the initial site i.e. are there secondary sites of infection and why do the bacteria hone in on these particular secondary sites.

Question 4

Bacterial Damage: do the bacteria cause any direct damage to the host (or is the damage fully attributable to the host response, as indicated below) and, if so, what is the nature of the bacterial damage. Can it be linked to any of the signs and symptoms in this case

The Immune Response Questions

Question 1

Host response: what elements of the innate and adaptive (humoral and cellular) immune response are involved in this infection.

Question 2

Host damage: what damage ensues to the host from the immune response?

Question 3

Bacterial evasion: how does the bacteria attempt to evade these host response elements.

Question 4

Outcome: is the bacteria completely removed, does the patient recover fully and is there immunity to future infections from this particular bacteria ?