Transcript Case_2_-_Question_2x

CASE 2
THE MICROBIOLOGY LABORATORY
Ashley Wang, Feb. 2016
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.
NARROWING IN ON THE
BACTERIAL CAUSES, WHAT
ARE THE MOST COMMON
BACTERIAL PATHOGENS
ASSOCIATED WITH THIS
INFECTIOUS SCENARIO.
Q1
Possible Bacterial Pathogens
STI
Associated Pathogen
Symptoms in Males
Chlamydia
Chlamydia trachomatis
Burning sensation during urination
Abnormal penile discharge
Scrotal inflammation
Epididymitis
Gonorrhea
Neisseria gonorrhoeae
Burning 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
Chlamydia trachomatis
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Size: 0.3-1µm in diameter
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Family: Chlamydiaceae
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Structure: Gram-negative
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Lifestyle: Obligate intracellular pathogen
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Cannot synthesize ATP, therefore rely on host cell for reproduction
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Life Cycle: Biphasic
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Elementary body: non-replicating and infectious
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Reticulate body: involved in replication and growth
Neisseria gonorrhoeae
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Size: 0.6-1µm in diameter
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Family: Neisseriaceae
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Structure: Gram-negative
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Usually seen in pairs
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Lifestyle: Obligate aerobe
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Oxygen is required, however can be anaerobically grown with an
appropriate electron acceptor
Natural Host: Human
WHAT SAMPLES ARE TAKEN
FOR LABORATORY TESTING
AND HOW IMPORTANT IS
THE MICROBIOLOGY
LABORATORY IN THE
DIAGNOSIS OF THIS DISEASE?
Q2
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)
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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
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
Nucleic Acid Amplification Test (NAAT)
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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
Gene Target
Roche Amplicor
Abbott LCx
Cytosine DNA
methyltransferase gene
Opacity protein genes
Amplification Technology PCR
LCR
Sensitivity
64.8 to 100%
88.2 to 97.3%
Specificity
93.9 to 100%
59.3 to 100%
Modified from Whiley, D.M. et al., 2006
NAAT
Advantages
 High sensitivity and specificity
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A single copy of the target genetic material can result in positive
signal
Easier sample collection
Faster results
Limitations
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Inability to detect antibiotic resistance
False-positive results
Commensal Neisseria species may acquire N. gonorrhoeae genes as a
result of frequent horizontal genetic exchange
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False-negative results
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Certain N. gonorrhoeae subtypes may lack the targeted sequence
Culture Tests – C. trachomatis
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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)
Less specific methods
thereby not recommended
by CDC
Modified from Elsevier. Murray:
Medical Microbiology 5e
Culture Tests – C. trachomatis
Advantages
 High specificity
 Antimicrobial susceptibility testing

Detect possible drug resistance
Limitations
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Relatively low sensitivity
Labour intensive
Long waiting time
Standardization problems
Strict sample transport requirements
Relatively high cost
Culture Tests – N. gonorrhoeae
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Inoculation of susceptible
cells with collected specimen
If specimens are non-sterile
They are streaked on selective
media (Thayer-Martin or MartinLewis)
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
Culture Tests – N. gonorrhoeae
Advantages
 Work with different types of specimens
 High specificity and sensitivity
 Isolates can be retained for
additional testing
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i.e. Drug resistance detection
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Low cost
Limitations
 Stringent sample transport requirements
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Viability of organisms must be maintained
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Relatively longer waiting time

A minimum of 24 – 72 hours is needed for a presumptive culture
report
Nucleic Acid Hybridization Tests
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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
Other Tests
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Nucleic acid genetic transformation tests
EIA Tests
DFA Tests
Serology Tests
Point-of Care Tests
FOR EACH POTENTIAL
PATHOGEN, WHAT ARE THE
EXPECTED RESULTS FROM
THESE TESTS.
Q4
C. trachomatis
Microscopic Examnation
• Visualization of chlamydial inclusions
after culturing the collected specimen
NAAT
• Detection of C. trachomatis by NAAT
N. gonorrhoeae
Suggestive Diagnosis
 Presence of urethral discharge
 Sexual exposure to a person carrying this bacteria
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
N. gonorrhoeae
Definitive Diagnosis
 Isolation of N. gonorrhoeae from sites of exposure

Demonstration of characteristic colonial morphology, gram-negative
morphology, and positive oxidase reaction
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Confirmation of isolates

Serology testing
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Nucleic acid testing
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Enzymatic testing
N. Gonorrhoeae
Ng, L.K., Martin, I.E., 2005
References
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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/
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.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.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.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/gettingtested#Overview1
References
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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.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.phacaspc.gc.ca.ezproxy.library.ubc.ca/lab-bio/res/psds-ftss/chlamydia-trachomatiseng.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-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.