Lab 7 Genital Tract Infections Flashcards

1
Q

Genital tract specimens are cultured to determine which diseases in females?

A

In females:
1. Cervicitis
2. (vulvo)-vaginitis
3. urethritis
4. salpingitis (pelvic inflammatory disease - PID)
5. Endometriosis
6. Genital ulcers
7. Bacterial vaginosis

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2
Q

Which diseases are genital tract specimens cultured for males?

A

In males:
1. Epididymitis
2. Urethritis
3. Prostatitis
4. Genital ulcers

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3
Q

How can genital infections affect newborns?

A

There are infections associated with childbirth and neonates from genital infections.

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4
Q

What is a common cause of cervicitis and urethritis that specimens are commonly sent to the lab for?

A

Chlamydia trachomatis
- collected to determine the presence of.

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5
Q

What is Neisseria gonorrhoea the causative agent of and is it a common specimen to send to the lab?

A

Neisseria gonorrhoeae, the agent of gonorrhea (‘GC’).

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6
Q

What affects the normal flora of the female genital tract?

A

Normal flora varies with age, hormonal levels, and pH.

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7
Q

What does the prepubescent (before menstruation) flora of the female genital tract?

A

Corynebacterium spp. (diphtheroid)
Coagulase negative Staphylococcus
Various anaerobes (skin like)

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8
Q

What is the reproductive age flora like and the organisms in female genital tract?

A

Reproductive age –> acid pH in genital tract of females.

Organisms include:
1. Lactobacilli
2. Corynebacterium spp.
3. Enterobacterales
4. Streptococci (including group B, should be reported in pregnancy)
5. Coag Neg Staph
6. S. aureus (unless its toxic shock syndrome)
7. Enterococcus spp.
8. Anaerobes
9. Yeast

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9
Q

What is the post-menopausal age normal flora like in females?

A

Fewer Lactobicilli
More Enterobacterales
may lack the yeast and mycoplasmas

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10
Q

What is the normal flora of the urethra for males?

A

The normal flora of the urethra includes relatively few skin organisms:
1. Coagulase-negative
staphylococci,
2. Corynebacterium spp,
3. Viridans streptococci.

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11
Q

What normal flora is on the external areas of the genital tract, the vulva and prepuce of the penis?

A

Both of the external areas of the genital tract- the vulva and the prepuce of the penis- may have:
1. Mycobacterium smegmatis,
2. other Gram positive bacteria and
3. some yeasts.

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12
Q

What condition/organism are routine genital specimens typically cultured for?

A

Routine genital specimens for culture are done basically just for GC, and in the majority of cases this is not even done by culture.

Candida albicans is a possible pathogen but in the last years diagnosis by culture has been replaced with observing yeast on a Gram-stained slide.

Trichomonas can be diagnosed with a wet mount, but its sensitivity is not too high and it gets higher using kits that detect surface antigens on the parasite and are ready in a few minutes.

GC = gonorrhea

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13
Q

What other methods (besides culturing) is used to diagnose GC (gonorrhea)?

A

Other methods:
- -> molecular-based/amplification

have replaced cultures for GC mostly, but some cultures are still received in labs. Selective media needs to be used for these.

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14
Q

Pre-analytically, what organisms are vaginal specimens for?

A

Vaginal specimens for:
Candida spp.
Trichomonas vaginalis
Bacterial vaginosis

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15
Q

Pre-analytically, what organisms are cervical specimens for?

A

Cervical specimens for:
Neisseria gonorrhoeae
Chlamydia trachomatis
Ureaplasma urealyticum

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16
Q

Pre-analytically, what organisms are urethral specimens (discharge - rarely asymptomatic) for?

A

Urethral specimens- discharge (rarely asymptomatic):
Staphylococcus aureus (more for wound-like specimens)
Beta-hemolytic streptococcus- Group A (uncommon)
Neisseria gonorrhoeae (also antigen detection)

Urethral specimens are also tested for the same pathogens as cervical specimens, with tests for Chlamydia trachomatis, Ureaplasma urealyticum, and Mycoplasma hominis found from most nongonococcal infections. (Exception- HPV)

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17
Q

External genitalia specimens- male and female
These infections are characterized by the presence of lesions or chancres, and include tests for:

A
  • Treponema pallidum (syphilis-chancre)- dark-field or fluorescent antibody preparations, serology
  • Haemophilus ducreyi (chancroid)-
  • HSV types 1 & 2 (herpes genitalis)- culture;
  • HPV (venereal warts) .

Candida is a pathogen to look for on external genitalia too.

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18
Q

What are reason that cause unacceptable genital tract specimens?

A

Unacceptable Specimens:
1. Specimen leaking
2. Improper container- specifically Neisseria gonorrhoeae loses viability easily and therefore should be sent in charcoal transport medium or specific test containers.
3. Clerical error (labeling, wrong info)
4. Specimen > 24 h old (depending on request & container)
5. Improper storage eg. refrigerated, frozen or left in transit at < 35ºC
6. Request for anaerobes
7. Lubricant used to collect
8. Vaginal specimen for GC or Chlamydia (should be cervix).
9. Do not accept vaginal swabs from women in childbearing years for “routine genital culture”. The disease or agent sought should be ordered specifically.

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19
Q

Why is a routine culture for genital tract specimens so limited (i.e. not a wide practice)? What is most often needed from the doctor?

A

“Because the agents of disease have diverse
culture and detection requirements
and selective media are often needed, a
“routine” genital culture, with the intent
to “detect what is there,” rarely has an indication. Occasionally such specimens are appropriate for prepubescent or postmenopausal women. Otherwise, laboratories
should provide a special requisition, such as in…, to be used to request the appropriate tests according to
the clinical condition and/or pathogen of interest.”

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20
Q

What media is used for Cervical / Urethral- Culture for GC?

A

Modified Thayer Martin (MTM), Chocolate Agar (CA)
incubated in CO2.

CA (non-selective is needed because there is a small number of GC (2%) that might be inhibited by MTM.

BA is also used in RRC lab for teaching purposes.

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21
Q

What media is used for Vaginal/ labial swab- for yeast?

A

(BA), SAB. Look for: Candida (on SAB & BA)= for vaginal specimens test on Gram smear and for external genitalia do culture.

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22
Q

Why might lack of isolation of a pathogen in the lab not indicate the pathogen is not the cause of the disease?

A

Many agents of disease are difficult to culture, and the lack of isolation may not indicate that the pathogen is not the cause of disease.

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23
Q

Why will a routine genital culture not detect presence of GBS? What is a better method?

A

Unless selective media and incubation are used, a routine genital culture will not detect carriage of GBS in all cases.

Methods that employ DNA detection (DNA probe or ‘PCR’) for the detection of GBS are more rapid than culture techniques.

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24
Q

What can cause false negative cultures?

A

False-negative cultures can result from contamination of the specimen with:

  1. Genital microbiota,
    especially E. faecalis, or
  2. From the inability to recognize non-hemolytic colonies (for GBS).
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25
Q

What is BV best diagnosed by?

What does BV stand for? Ask Mariella, guess Bacterial vaginosis

A

Because of the difficulty in evaluating the significance of G. vaginalis in culture, unless it is clearly predominant and numerous, BV is best diagnosed by Gram stain (Nugent score) or/and in the Physician’s office (sniff test)

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26
Q

Why is more than one confirmatory method is required for ID of N. gonorrhoeae in the case of child abuse?

A

More than one confirmatory method for identification of N. gonorrhoeae is essential for potential cases of child abuse, since nearly every method has some errors.

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27
Q

For what culture/condition is unscented white wax candles used for and why?

A

If using candle jars to generate a CO2 atmosphere, use only unscented white wax candles. Colored candles yield toxic substances for GC.

GC = gonorrhea

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28
Q

Describe the expected colony morphology on CA plates for Neisseria Gonorrhoeae (GC)? Gram stain?

A

Small, translucent, gray and mucoid colonies.

Gram stain: g-dc

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29
Q

Once you have a suspected N. gonorrhoeae (GC) colony what tests are done to confirm it?

A

Gram stain
30% H2O2 (not in our lab)
Growth in basic media
API NP to confirm ID.

30
Q

If you have no growth detected, now long do you incubate plates before reporting ‘No growth’?

A

72 hours for GC

31
Q

What direct smear result is reported right away to the Dr. and is considered diagnostic?

A

PMN’s with g-dc inside them (i.e. intracellular).

32
Q

We perform API NH to confirm ID of N. gonorrhoeae (GC) what is needed in clinical labs?

A

A second confirmatory test is needed for GC due to the nature of the reporting, especially from non-genital sites and from children (e.g. sexual abuse, etc.).

33
Q

Why does a negative culture not rule out infection for N. gonorrhoeae (GC)?

A

Because of its labile nature of N. gonorrhoeae.

34
Q

What should be done if no GC was found after 72 hours of incubating?

A

Plates should be flooded with oxidase reagent. If purple color shows, colonies should be subbed to a fresh CA plate and investigation continues.

35
Q

What conditions in pregnant women is Streptococcus agalactiae associated with?

A

Asymptomatic bacteriuria
Urinary tract infection
Amnionitis

Women who have recently delivered:
- Endometritis
- Wound infection

Baby:
Early -onset neonatal disease (1st week)

36
Q

What can be given to prevent infection in pregnant mom’s from S. agalactiae?

A

Intrapartum ampicillin prophylaxis (or penicillin)

Given to women with positive prenatal cultures and certain perinatal risk factors.

37
Q

When is a specimen collected for screening of S. agalactiae on pregnant women?

A

35-37 weeks gestation

Swab inserted in lower vagina (no speculum should be used) and then in the rectum.

38
Q

How should the S. agalactiae swab be transported to the lab?

A

In transport media, sent to lab ASAP, and ideally planted within 24 hours.

39
Q

What do labs also use to increase the chances of finding Strep B. on pregnant women?

A

Selective/enrichment broth

Subbed to 2ndary BA plate next day, and treated like primary plate.

40
Q

How do you plant from the screening swab for pregnant women to look for Strep. B?

A

Rotate swab over an area about the size of a loonie. Rotate swab to ensure recovery of unevenly distributed pathogen that may be on swab.

Streak plates for isolation.

41
Q

What colonial morphology are you looking for on the BA plates for Strep B?

A

med-lg, gy, translucent colonies with a small zone of B-hem or no zone of hem.

Qty is irrelevant.

42
Q

What follow-up tests are done on suspected Strep B colonies?

A

Perform catalase (neg)
Gram stain if catalase neg.
–> look for g+c in pairs and chains
Finally one of the following tests:
CAMP test (pos)
Lancefield (pos for group B)

43
Q

Does a positive culture of Strep B always indicate infection?

A

No, just colonization. The person may or may not have an infection.

44
Q

How do you report for Strep B?

A

Report as: Streptococcus agalactiae-Group B isolated. “Intra-partum antimicrobic therapy of choice is penicillin or ampicillin. Please notify the Microbiology lab immediately if patient is penicillin allergic so alternate antimicrobic testing may be performed.”
The absence of any pathogens requested should be specified:
“No Group B streptococci isolated”

45
Q

Why may PCR testing method be used instead of culture? But what is the downside?

A

Real-time PCR is more sensitive and fast than culture but the organism will not be available for AST if needed.

46
Q

What can cause false negative cultures?

A
  1. False-negative cultures can result from contamination of the specimen with genital microbiota, especially E. faecalis, or from the inability to recognize nonhemolytic colonies of Strep agalactiae.
  2. Listeria colonies can be confused with S. agalactiae if the Gram stain or catalase
    test is omitted.
47
Q

What population does bacterial vaginosis occur in?

A

Bacterial vaginosis (BV) is a clinical syndrome in women during their childbearing years characterized by an abnormal vaginal discharge. It is normally accompanied by a rise in pH from 4.5 and an amine (fishy) smell, especially after addition of KOH to the discharge.

48
Q

What type of cells is observed in a smear with bacterial vaginosis (BV)?

A

Clue cells, vaginal SECs coated with bacteria such that the cell borders are obliterated, may also be observed.

49
Q

If a women has BV, what obstetric conditions are they at risk for?

A

Low birth weight
Premature delivery
Associated with preterm birth, miscarriage, amniotic infections, and postpartum endometritis.

50
Q

What organisms does the microbiota of the vagina shift to in BV?

A

Shifts from predominant Lactobacilli to a mixture of:
- Gardnerella vaginalis
- Prevotella spp.
- Mobiluncus spp.
- other anaerobes
- Mycoplasma hominis (may also be present)

Appear as small gram-variable pleomorphic bacilli.

51
Q

What do the bacteria mixture in BV looks like in a direct gram stain?

A

G. vaginalis organisms appear as small Gram-variable pleomorphic bacilli, varying from coccobacilli to longer forms up to 2 to 3 µm; cells can palisade or appear coryneform and irregularly shaped.

Mobiluncus spp. are curved Gram-negative rods.

52
Q

What is the Nugent scoring system?

A

The comparison of relative numbers of lactobacilli (medium to large Gram-positive rods) with those of Gram-negative curved rods and Gram-variable coccobacilli allows for the diagnosis of BV to be made, using the Nugent scoring system.

See table for the scores. They are added together for a total score and then interpreted.

Score 7-10 = BV pos
Score 4-5 = BV indeterminate
Score 0-3 = BV neg

53
Q

What type of specimen is gathered for Bacterial Vaginosis (BV)?

A

Vaginal secretions from the posterior fornix using sterile cotton or Dacron swab. Transport in Amies’ media.

54
Q

How do you report for Bacterial vaginosis?

A

You report according to the scores as follows:

0 to 3, “Morphotypes consistent with normal vaginal microbiota”;

4 to 6, “Mixed morphotypes consistent with transition from normal vaginal microbiota”;

7 to 10, “Mixed morphotypes consistent with bacterial vaginosis.”

55
Q

What lab method is more sensitive for yeast infections - gram stain or culture?

A

Vaginal culture for yeasts is more sensitive than gram stain.

May be used for women with consistent symptoms whose wet mount exams or gram smears are negative.

56
Q

How is Candidiasis clinically characterized as?

A

White discharge and a normal pH below 4.2

57
Q

What does it signify when yeast is seen on the gram stain?

A

Means the amount of yeast is significant. Report and the Dr. will make a decision together with the clinical symptoms.

58
Q

What amount is considered suspicious growth on a yeast culture?

A

3+ to 4+

59
Q

What is the colony morphology for Candida albicans you are looking for on BA and SAB plates?

A
  • BA morphology – sm white opaque, pasty/dry with or without “feet” (feathery or lacy peripheral fringe around the colony. This occurs most commonly on serum-enriched media, such as sheep blood agar or chocolate).
    • SAB morphology – white, creamy or pasty colonies
60
Q

What indicates yeast on a wet mount or gram stain?

A

Wet mount: “budding yeast” or Gram stain: Yeast-like cells (purple)

61
Q

What is the germ tube test?

A

Rabbit plasma - 2 hr @ 35C with yeast.

62
Q

What indicates the presence of Candida albicans on a germ tube test?

A

G.T. defined as: A filamentous extension from a yeast cell that is about ½ the width x 3-4 times the length, showing no constriction at the point of origin (parallel sides)

  • A good rule is to see at least five germ tubes before calling an organism positive, as rare tubes may be produced by other species.
  • For this body site, we will consider Germ tube positive yeasts as Candida albicans. If Germ tube is negative: Candida spp.
63
Q

Why is a positive germ tube test only considered presumptive?

A

5% of C. albicans are GT Θ therefore, G.T. test should be presumptive. (C. tropicalis & C. stellatoides can also be GT )

64
Q

What is the best medium (agar) to isolate yeast for the germ tube test?

A

Sabouraud dextrose agar

BA is an acceptable substitute.

65
Q

What could cause a false positive for Candida albicans if one is not careful?

A

Some C. tropicalis isolates produce pseudohyphae and some also produce germ tubes that require careful observation to discriminate from those of C.albicans.

66
Q

What is another diagnostic criteria for C. albicans (although not generally performed from genital tract specimens)?

A

Production of Chlamydospores is diagnostic for C. albicans (not generally performed from genital tract specimens)

67
Q

What other test could be done for C. albicans besides culture, wet mount and a gram stain?

A

Other tests for yeasts include:
CHROMagar= Presumptive – typical growth C. albicans screen – a preformed enzyme test kit which detects β-galactosaminidase (+) and L-proline aminopeptidase (-).

68
Q

What tests in the lab can provide a definitive ID for C. albicans?

A

Definitive ID:
* Chlamydospore production
* API AUX: Assimilation and Fermentation tests
* Preformed enzymes
* Vitek yeast card
* API Rapid Yeast ID
* Maldi-Tof

69
Q

How is assimilation and fermentation used in API AUX to ID C. albicans? Limitations?

A

Assimilation is the ability to use a sole carbon substrate for growth.
Fermentation is indicated by turbidity, change of color or the indicator and gas production (Durham tube)

Limitations A false-positive can occur in the carbohydrate fermentation tests due to endogenous carbohydrates present in the yeast cells if a large inoculum is used.

70
Q

What type of vaginal discharge characterizes Trichomonas vaginalis?

A

Yellowish-green, foul-smelling discharge containing >= 30 WBC per high power field.

71
Q

What has been traditionally used to detect T. vaginalis in the past and why is it no longer used?

A

Wet-mount examination for detection of T. vaginalis has been used in the past, but has a low sensitivity, particularly when specimens are delayed in transit. Wet mount without Trichomonas culture may miss 50% of cases.

Most labs used to have a culture broth for Trichomonas. They have to be incubated for a few days and observed with a wet mount regularly, which is labour intensive and takes a long time to report.

Culture also requires maintenance of specialized media.

72
Q

What tests are now used instead of wet mount and gram stain for Trichomonas vaginalis?

A

Trichomonas rapid antigen and nucleic acid amplification tests (NAT) are available
and have greater sensitivity for detecting infection.

In our lab we use the OSOM test.