1. عفونت هاي دستگاه تناسلي: روش صحيح نمونه گيري، كشت و تفسير
دكتر داريوش شكري
آزمايشگاه ميکروب شناسی بيمارستان الزهرا (س) اصفهان

2. Genital collection for:
Patients in high risk situations:
Patients known to have gonorrhea
Females with mucopurulent cervicitis, urethral syndrome, endometriosis, and salpingitis
Neonates born to infected mothers
Infertility investigations

3. Sexually active asymptomatic females:
age 25 years or younger
pregnant
evidence of purulent or mucopurulent cervical discharge
Exhibit endocervical bleeding, induced by swabbing on examination
Have had a new sex partner in the preceding 2 months
Use no contraceptives or a non-barrier method for contraception

4. Collection, Transport and Culture

5. PROCEDURE: 1. MALE: A. Urethral specimens should not be collected until AT LEAST one hour AFTER urination. B. Urethral discharge can be collected on a swab C. If no discharge is obtained, a Sterile Rayon Tipped or Dacron Swab, should be inserted into the distal urethra for approx. 2-4 cm. and gently rotated for 2 to 3 seconds.

6. For Females
Cervical specimens should be collected after removing excess mucous from the cervical os and surrounding mucosa
Use a second swab to
collect specimen by
rotating the swab for
10 to 30 secs. in the
endocervical canal
Collect vaginal specimens
using a speculum without
any lubricant

7. GENITAL TRACT—'GC' ONLY CULTURES
PROCEDURE NOTES:
Cotton swabs contain fatty acids that can be inhibitory to gonococci.
GC (gonococci) can die quickly. So IMMEDIATE transport to the lab is important so that the conditions for viability of the gonococci can be met.
Record of time collection is needed

8. Vaginal specimens are NOT acceptable for the detection of N
Vaginal specimens are NOT acceptable for the detection of N. gonorrhoeae.

9. TRANSPORT AND STORAGE: 1. Take sample to lab IMMEDIATELY. 2
TRANSPORT AND STORAGE: 1. Take sample to lab IMMEDIATELY. 2. Store at room temperature 3. Fill out Microbiology Request Form. Include TIME of collection.

10. GENITAL TRACT CULTURES
GENITAL TRACT—ROUTINE CULTURES PROCEDURE NOTES: Routine cultures include detection of most common pathogens such as Yeast, N. gonorrhoeae, Group B streptococci, Gardnerella vaginalis, and any other predominant organism not considered usual flora.

11. Genital Specimens Specimen source Potential Pathogens
Primary plating media
Special considerations
Cervix
Chlamydia; GC; herpes
SBA, Choc, TM; viral transport media for herpes
NAT testing recommended for GC, CT
Cul-de-sac
Anaerobes, GC, CT, enterics
SBA, choc, TM,
Mac, ana, thio
Collect aspirate;
Anaerobe transport
Endometrium
Mixed aerobes /anaerobes
Surgical biopsy or sheathed catheter
Vagina
Group B strep;
Mixed aerobes anaerobes BV
SBA; LIM or other special broth
Culture for Group B strep only; do not culture for BV

12. Culture Setup
1. Cervical/vaginal/urethral cultures - specimens are set up on the following media: a. BAP b. MAC agar (or EMB) c. Chocolate agar d. BAP anaerobicaly e. Thayer Martin agar BHI may useful for recovery of few microorganism B. Incubate media 1. Temperature: 35ºC 2. Atmosphere: BAP, CHOC, TM - CO2, MAC - ambient air 3. Time: hours

13. Genital Tract Pathogens and Specimen Type
Organism
Specimen Type
Neisseria gonorrhoeae
Cervical, urethral, anal, vaginal swabs.
Bacteria
Prostatic fluid, cervical, vaginal.
Trichomonas vaginalis
Vaginal, prostatic fluid.
Fungi
Anal, vaginal or cervical.
Anaerobes
Epididymis aspirate, amniotic fluid, abscess.
HSV
Genital or perianal lesion.
Chlamydia trachomatis
Rectal, cervical, urethral, bubo or ulcer material.
Haemophilus ducreyi
Material from ulcers (genitalia and perianal) and from inguinal nodes.

14. D. Normal flora
1. Urethral a. Coagulase-negative staphylococcus b. Diptheroids (Corynebacteria species) c. Various anaerobes 2. Vulva and penis a. Mycobacterium smegmatis b. Other gram positive bacteria

15. 3. Cervix and Vaginal a. Lactobacillus sp
3. Cervix and Vaginal a. Lactobacillus sp. (predominant organism found in healthy vaginal specimens) b. Staphylococcus species c. Diphtheroids d. Microaerophilic and anaerobic cocci e. Anaerobic gram-negative rods f. Streptococcus species including Enterococcus species g. Clostridium species h. Enterics i. Group B streptococcus

16. Culture Interpretation
A. Quantities, identify and perform sensitivities on all potential pathogens. B. Quantities and report normal flora organisms as a whole without sensitivities. All organisms will be quantitated and reported as “Normal vaginal flora.” C. Hold all plates for minimum of 48 hours before sending out the report. Cultures for Neisseria gonorrhoeae should be held 72 hours.

17. Neisseria gonorrhoeae

18. nonmotile, cocci (diplo) gram negative
No spores formation
most species grow optimally at 35 to 37°C.
capnophilic
grow best in a moist environment
N. gonorrhoeae is always considered a pathogen, regardless of the site of isolation.

19. Direct smear
When properly performed, the Gram stain has a sensitivity of 90-95% and a specificity of % for diagnosing genital gonorrhea in symptomatic men
Gram-stained smears of endocervical specimens have a sensitivity of 50-70%, depending on the adequacy of the specimen and the patient population.

20. For smear preparation, the swab is rolled gently over the surface of a glass slide in one direction only.
Smears prepared from specimens submitted in transport media (e.g., charcoal). may be more difficult to interpret because of distortion of the PMNs or to interfering substances

21. Direct smear

22. media for culture
A variety of enriched selective media for culture of N.gonorrhoeae are available :
Modified Thayer-Martin (MTM) medium,
Martin-Lewis (ML) medium,
GCLect medium,
New York City (NYC) medium.
MTM , ML, GCLect are chocolate agar-based media that are supplemented with growth factors

23. The CO2 level of the incubator should be 3-7%;
Higher CO2 concentrations may actually inhibit growth of some strains.

24. candles candle jars should be made of white wax or bees' wax;
Plates are inspected at 24, 48, and 72 hours before a final report of "no growth" is issued.
Suspect colonies are subcultured to chocolate agar, incubated, and used as an inoculum for identification procedures.

26. chocolate agar media is the best routine medium for this bacteria if it base be good and be in QC.

27. oxidase + and catalase +

28. Some other bacteria may also grow on selective media.
These organisms include:
Kingella denitrificans
Moraxella species (other than M. catarhalis),
Acinetobacter species,
Capnocytophaga species.

29. K. denitrificans grows well on MTM medium and produces colony types that resemble those of N.gonorrhoeae
Catalase test is useful in presumptively identifying gonococci and;
K. denitrificans : negative catalase reaction

30. Acinetobacter species can be differentiated by their negative oxidase reaction
Capnocytophga species are both oxidase and catalase negative.

31. M. catarrhalis and other Moraxella species, like gonococci, are oxidase + and catalase +
Dnase test is positive for M. catarrhalis

32. Carbohydrate-Utilization Tests
Cystine-tryptic digest semisolid agar-base (CTA) medium containing 1% carbohydrate and a phenol red pH indicator
The usual test series includes CTA-glucose, -maltose, -sucrose, and -lactose, plus a carbohydrate-free CTA control.
CTA media are inoculated with a dense suspension of the organism from a pure 18- to 24-hour culture on chocolate agar.

33. The inoculum is restricted to the top 1/2 inch of the agar-deep tubes.
The tubes are incubated in a non-CO2 incubator at 35°C

35. Haemophilus ducreyi
is a fastidious gram-negative coccobacillus causing the sexually transmitted disease chancroid
It is a major cause of genital ulceration in developing countries characterized by painful sores on the genitalia.
Another early symptom is dark or light green shears in excrement. Chancroid starts as an erythematous papular lesion that breaks down into a painful bleeding ulcer with a necrotic base and ragged edge.

36. On a global basis, chancroid is thought to be the most common cause of genital ulcer disease (GUD)
Other causes of GUD include Treponema pallidum, Chlamydia trachomatis serovars L1, L2 and L3, Calymmatobacterium granulomatis and herpes simplex virus

37. H. ducreyi can be cultured on chocolate agar.
H. ducreyi gram stain appear as "school of fish."

38. the swab in transport medium (eg, Amies or Amies with charcoal) needs to reach the laboratory within 4 h because H. ducreyi will not survive well beyond this time.
All inoculated media should be incubated in 5% CO2 at 33°C to 35°C (it is critical that the temperature does not exceed 35°C) in a humid environment.

40. additional identification tests to consider include:
oxidase (positive for H. ducreyi)
catalase (negative for H. ducreyi)
X factor nutritional requirement (H.ducreyi requires X factor for growth.

41. BACTERIAL VAGINOSIS (BV)
BV is the most common cause of vaginitis symptoms among women of childbearing age.
Also known as “nonspecific vaginitis” or “Gardnerella-associated vaginitis”,
BV is associated with sexual activity.

42. Who Should be Screened for BV?
Women with vaginal symptoms
esp. if failed therapy
Pregnant women at high risk of preterm birth
Pregnant women with genital symptoms
rule out trichomoniasis as well
Women with gynecologic surgery
Bacteria may be detected on a Pap smear.
• Alkaline pH (greater than 4.5)

43. Sample of vaginal discharge is looked at under the microscope for appearance of “clue” cells.
A couple drops of potassium hydroxide are mixed with the sample. The mixture gives off a fishy odor if gardnerella is present.
Vaginal culture is taken. A sample is grown in the laboratory and identified. Results take 3 working days.

44. Clue Cell of Bacterial vaginosis (BV)

45. The presence of Gardnerella vaginalis on culture can not be used to diagnose BV, since it is present in approximately 20-40% (up to 50 %) of healthy women. 

46. Gardnerella vaginalis grows as small, circular, convex, gray colonies on chocolate agar.
Gram stain is the method of choice for diagnosis of  bacterial vaginosis.
Gardnerella is a small, gram- positive to gram-variable staining rod shape.
Catalase negative
Beta hemolytic in human blood agar

47. It has a gram-positive cell wall, but because the cell wall is so thin it can appear either gram-positive or gram-negative under the microscope. It is associated microscopically with clue cells, which are epithelial cells covered in bacteria

49. Trichomonas vaginalis
Common sexually transmitted disease
Disease associations and adverse outcomes
Vaginitis
Urethritis—men and women
Outcomes
Adverse pregnancy events
Associated with increased HIV shedding

50. Trichomonas vaginalis Diagnosis
Culture- “gold standard”
Diamond’s media
InPouch TV; BioMed Diagnostics, San Jose CA)
Barenfanger J, et. al J Clin Microbiol 40:1387.
Wet mount—insensitive (~ 50%)
Rapid tests
XenoStrip-Tv (GenzymeDiagnostics, Inc. San Antonio, Tex.)
more sensitive than wet prep
less sensitive than culture
useful as a POC test

51. Lab detection GLC—no longer used
Detection of sialidases (neuraminidases that remove sialic acid from sialogly-coconjugates)
In BV, associated with Prevotella and Bacteroides sp.
Colorimetric test BV Blue System (Gryphus Diagnostics—91.7% sensitive; 97.8% specific

52. Chlamydia Chlamydia tesing is available by three methods:
culture, direct flourescent antibody (DFA) or molecular probe.
The culture method can be used for all specimen sites and is the only acceptable method for diagnosis of chlamydia in children.

53. Chlamydia Culture Collection
1 Utilizing a sterile swab, obtain a suitable specimen. 2 Place the swab in transport media (M4 media tube with pink liquid). 3 Label the transport media vial. 4 Transport the vial to the laboratory (may be refrigerated).

54. For HSV lesions Fluid from lesions should be aspirated using a syringe
Swab can be used to collect vesicle fluid or cellular material from the base of the lesion before crusting and healing have begun

55. Thanks