1. CASE 2 THE MICROBIOLOGY LABORATORY Ashley Wang, Feb. 2016

2. The Case 21-year-old Naser G. recently hooked up with a new sexual partner. This morning he noticed a burning pain in his penis during urination followed by a greenish discharge. He immediately goes to the student health clinic. The clinic doctor asks Naser about his recent sexual history and he recounts how he had unprotected sexual intercourse with a new partner about one week ago. The new partner claimed that she did not have any sexually transmitted infections. The doctor asks Naser to provide a urine sample to send to the Microbiology Laboratory. The doctor prescribes antibiotics for him and counsels him on safe sex practices and on the importance of encouraging his new partner to come in for testing too.

3. NARROWING IN ON THE BACTERIAL CAUSES, WHAT ARE THE MOST COMMON BACTERIAL PATHOGENS ASSOCIATED WITH THIS INFECTIOUS SCENARIO. Q1

4. Possible Bacterial Pathogens STIAssociated PathogenSymptoms in Males ChlamydiaChlamydia trachomatisBurning sensation during urination Abnormal penile discharge Scrotal inflammation Epididymitis GonorrheaNeisseria gonorrhoeaeBurning sensation during urination Thick white or yellow-green discharge Inflammation of the urethra Nongonococcal Urethritis (NGU) Chlamydia trachomatis (most common); Ureaplasma urealyticum Haemophilus vaginalis Mycoplasma gentitalium Burning sensation during urination Thin penile discharge

5. Chlamydia trachomatis  Size: 0.3-1µm in diameter  Family: Chlamydiaceae  Structure: Gram-negative  Lifestyle: Obligate intracellular pathogen  Cannot synthesize ATP, therefore rely on host cell for reproduction  Life Cycle: Biphasic  Elementary body: non-replicating and infectious  Reticulate body: involved in replication and growth

6. Neisseria gonorrhoeae  Size: 0.6-1µm in diameter  Family: Neisseriaceae  Structure: Gram-negative  Usually seen in pairs  Lifestyle: Obligate aerobe  Oxygen is required, however can be anaerobically grown with an appropriate electron acceptor  Natural Host: Human

7. WHAT SAMPLES ARE TAKEN FOR LABORATORY TESTING AND HOW IMPORTANT IS THE MICROBIOLOGY LABORATORY IN THE DIAGNOSIS OF THIS DISEASE? Q2

8. Diagnosis of Disease  In this case, a urine sample is taken for testing Alternative methods include  Blood tests  A swab inside the penis (commonly used in the old days)  Urine test is important to the diagnosis of STD because:  Many STDs share similar symptoms or show no symptoms  Early detection and treatment is important in preventing complications

9. EXPLAIN THE TESTS THAT WILL BE PERFORMED ON THE SAMPLES IN ORDER TO DETECT (ALL OF) THE BACTERIAL PATHOGENS THAT MAY BE CAUSING THIS DISEASE. Q3

10. Nucleic Acid Amplification Test (NAAT)  Able to identify bacteria by amplifying its DNA  No viable organisms required  Can be performed on swab specimen or urine samples  Commonly used for detection of chlamydia and ghonorrhea Overview of Commercial N. gonorrhoeae NAATs Roche AmplicorAbbott LCx Gene TargetCytosine DNA methyltransferase gene Opacity protein genes Amplification TechnologyPCRLCR Sensitivity64.8 to 100%88.2 to 97.3% Specificity93.9 to 100%59.3 to 100% Modified from Whiley, D.M. et al., 2006

11. NAAT Advantages  High sensitivity and specificity  A single copy of the target genetic material can result in positive signal  Easier sample collection  Faster results Limitations  Inability to detect antibiotic resistance  False-positive results  Commensal Neisseria species may acquire N. gonorrhoeae genes as a result of frequent horizontal genetic exchange  False-negative results  Certain N. gonorrhoeae subtypes may lack the targeted sequence

12. Culture Tests – C. trachomatis  Inoculation of susceptible cells with collected specimen  Intracytoplasmic inclusions develop on infected cells  They can be detected by  a fluorescein-conjugated monoclonal antibody that binds to the major outer membrane protein (MOMP)  EIA  Iodine  Giemsa C. Trachomatis is grown in cell cultures and detected by staining inclusion bodies (arrows) Modified from Elsevier. Murray: Medical Microbiology 5e } Less specific methods thereby not recommended by CDC

13. Culture Tests – C. trachomatis Advantages  High specificity  Antimicrobial susceptibility testing  Detect possible drug resistance Limitations  Relatively low sensitivity  Labour intensive  Long waiting time  Standardization problems  Strict sample transport requirements  Relatively high cost

14. Culture Tests – N. gonorrhoeae  Inoculation of susceptible cells with collected specimen  If specimens are non-sterile  They are streaked on selective media (Thayer-Martin or Martin- Lewis)  Media contain antimicrobials that inhibit the growth of other bacteria  If specimens are sterile  They are streaked on non-selective media (chocolate agar)  Organisms other than N. gonorrhoeae can grow as well Pathogen Profile Dictionary

15. Culture Tests – N. gonorrhoeae Advantages  Work with different types of specimens  High specificity and sensitivity  Isolates can be retained for additional testing  i.e. Drug resistance detection  Low cost Limitations  Stringent sample transport requirements  Viability of organisms must be maintained  Relatively longer waiting time  A minimum of 24 – 72 hours is needed for a presumptive culture report

16. Nucleic Acid Hybridization Tests  Used to detect C. trachomatis or N. gonorrhoeae  Two FDA-proved nucleic acid hybridization assays are available  Gen-Probe PACE 2  Digene Hybrid Capture II  If result is positive, then organism-specific tests need to be followed by  Assays do not differentiate between the two pathogens One Major Advantage  Specimens can be stored/transported without refrigeration for up to 7 days

17. Other Tests  Nucleic acid genetic transformation tests  EIA Tests  DFA Tests  Serology Tests  Point-of Care Tests

18. FOR EACH POTENTIAL PATHOGEN, WHAT ARE THE EXPECTED RESULTS FROM THESE TESTS. Q4

19. C. trachomatis Microscopic Examnation Visualization of chlamydial inclusions after culturing the collected specimen NAAT Detection of C. trachomatis by NAAT

20. N. gonorrhoeae Suggestive Diagnosis  Presence of urethral discharge  Sexual exposure to a person carrying this bacteria

21. N. gonorrhoeae Presumptive Diagnosis Microscopic Examnation Gram-negative diplococci detected from a smear of urethral discharge Oxidase Test Development of gram-negative, oxidase-positive diplococcus Non-culture Laboratory Tests Detection of N. gonorrhoeae by NAAT or nucleic acid probe test

22. N. gonorrhoeae Definitive Diagnosis  Isolation of N. gonorrhoeae from sites of exposure  Demonstration of characteristic colonial morphology, gram-negative morphology, and positive oxidase reaction  Confirmation of isolates  Serology testing  Nucleic acid testing  Enzymatic testing

23. N. Gonorrhoeae Ng, L.K., Martin, I.E., 2005

24. References  American Association for Clinical Chemistry. (2014). Chlamydia and gonorrhea NAAT screening method endorsed by CDC. Retrieved Feb. 6, 2016, from https://labtestsonline.org/news/140521naat/https://labtestsonline.org/news/140521naat/  CDC. Centers for Disease Control and Prevention. (2013). Characteristics of N. gonorrhoeae and related species of human origin. Retrieved Feb. 6, 2016, fromhttp://www.cdc.gov/std/gonorrhea/lab/ngon.htmhttp://www.cdc.gov/std/gonorrhea/lab/ngon.htm  CDC. Centers for Disease Control and Prevention. (2014). Recommendations for the laboratory-based detection of chlamydia trachomatis and neisseria gonorrhoeae — 2014. Retrieved Feb. 6, 2016, from http://www.cdc.gov/mmwr/preview/mmwrhtml/rr6302a1.htmhttp://www.cdc.gov/mmwr/preview/mmwrhtml/rr6302a1.htm  CDC. Centers for Disease Control and Prevention. (2014). Tests to detect C. trachomatis and N. gonorrhoeae. Retrieved Feb. 6, 2016, fromhttp://www.cdc.gov/std/laboratory/2014LabRec/recommendations.htmhttp://www.cdc.gov/std/laboratory/2014LabRec/recommendations.htm  Chernesky, M. A. (2005). The laboratory diagnosis of chlamydia trachomatis infections. The Canadian Journal of Infectious Diseases & Medical Microbiology, 16(1), 39-44.  Cook, R.L. et al. (2005) "Systematic Review: Noninvasive Testing for Chlamydia trachomatis and Neisseria gonorrhoeae" Ann Intern Med. 142:914-925.  Healthline Editorial Team. (2014). STD testing. Retrieved Feb. 5, 2016, from http://www.healthline.com/health/sexually-transmitted-diseases/getting- tested#Overview1http://www.healthline.com/health/sexually-transmitted-diseases/getting- tested#Overview1

25. References  Johnson, R. E., et al. (2002). Screening tests to detect chlamydia trachomatis and neisseria gonorrhoeaeinfections --- 2002. Retrieved Feb. 3, 2016, fromhttp://www.cdc.gov/mmwr/preview/mmwrhtml/rr5115a1.htmhttp://www.cdc.gov/mmwr/preview/mmwrhtml/rr5115a1.htm  Ng, L. K., & Martin, I. E. (2005). The laboratory diagnosis of neisseria gonorrhoeae. The Canadian Journal of Infectious Diseases & Medical Microbiology, 16(1), 15-25.  Public Health Agency of Canada. (2012). Pathogen safety data sheet - infectious substances. Retrieved Feb. 4, 2016, from http://www.phac- aspc.gc.ca.ezproxy.library.ubc.ca/lab-bio/res/psds-ftss/chlamydia-trachomatis- eng.phphttp://www.phac- aspc.gc.ca.ezproxy.library.ubc.ca/lab-bio/res/psds-ftss/chlamydia-trachomatis- eng.php  Shakespeare, M. (2002). Zoonoses Pharmaceutical Press.  Spence, J. M., Wright, L., & Clark, V. L. (2008). Laboratory maintenance of neisseria gonorrhoeae. Current Protocols in Microbiology, Chapter 4, Unit 4A.1.  University of California, Santa Barbara. (2016). STI symptom chart. Retrieved Feb. 3, 2016, from http://www.soc.ucsb.edu/sexinfo/article/sti-symptom-charthttp://www.soc.ucsb.edu/sexinfo/article/sti-symptom-chart  Whiley, D. M., Tapsall, J. W., & Sloots, T. P. (2006). Nucleic acid amplification testing for neisseria gonorrhoeae : An ongoing challenge. The Journal of Molecular Diagnostics : JMD, 8(1), 3-15.