1. MICROBIOLOGY PRACTICAL
Reproductive block
2014

2. Sexually Transmitted Diseases Practical
Prof kambal
Dr.Ali M Somily
Dr.Malak El-Hazmi

3. Objectives
Name various etiological agents causing sexually transmitted diseases
Describe the clinical presentations of sexually transmitted diseases
Discuss the microbiological and no microbiological methods for diagnosis of sexually transmitted diseases
Outline the management of sexually transmitted diseases .

5. Ureaplasma urealyticum 3- Fungus Yeast – Candida albicans 4- Parasites
Four Classes of STD’s
1- Viruses-
Herps
HIV
Hepatitis B
Human papilloma virus
2- Bacterial
Syphilis
Gonorrhea
Chlamydia
Chancroid
Mycoplasma hominis
Ureaplasma urealyticum
3- Fungus
Yeast – Candida albicans
4- Parasites
Trichomonas
Ectoparasites
Phithrus pubis - Pubic louse
Sarcoptes scabiei

7. Bacterial STDs

8. Case 1
A 23-year-old alcoholic and drugs (cocaine) addict single male arrived from his trip to South East Asia six months ago. He gave history of multiple sexual partners. Two months ago he developed ulcer on his penis which disappeared compeletly. A full physical notes a rash on both her palms and her soles

9. ? What are the possible causes for his presentation
Treponema Pallidum
Herpes Simplex Virus 2
Haemophilus Ducreyi

10. How could you differentiate between them based on s/s of the patient?
Ulcer
Etiology
Lymphadenopathy (Babo)
Systemic
Chancroid
Haemophilus Ducreyi
Wet , painful
Inguinal tender
Present
Chancer
Treponema Pallidum
Dry, painless and raised margin
Inguinal part of generalize disease
Depends on stage
Ulcerated Vesicles
Herpes Simplex Virus 2
Multiple shallow painful
Occasionally present
In primary

11. Syphilis
syphilis is a disease caused by a corkscrew-shaped bacterium (a spirochete) called Treponema pallidum. It causes disease when it penetrates broken skin of the genitals or the mucous membranes of the mouth or anus.
It can also be passed from an infected person through an open cut or wound and from mother to child.
Symptoms: 3-staged disease

13. Syphilis - Primary syphilis
Painless penile chancre in primary syphilis due to Treponema pallidum:
primary syphilis infection vagina ulcers

14. The lesion are sampled and identified through
dark-field microscopy

15. Syphilis - 2nd stage - or secondary syphilis .
This stage occurs in about 6-8 weeks after formation of sore if the chancre is not marked and the patient untreated
Typically, general signs appear: fever, the aches , headache , a tiredness , a rash as well as lesions on the palms of the hands and soles (feet), glossitis (lesions in the tongue), the lesions contain the bacteria, they are highly contagious

16. secondary syphilis. B A
Mucous patches (A and B) are superficial mucosal erosions seen in secondary syphilis. They can be found on any mucosal surface and are highly infectious

17. Base on the finding, what is the most likely diagnosis
Base on the finding, what is the most likely diagnosis? Briefly outline the management of this patient?
Dx;
Primary syphilis
Rx;
Benzathine penicillin IM---allergy Doxycycline
Patient Counseling and Education
He should be tested for other STD especially HIV

18. Syphilis - 3rd stage or Tertiary / Acute stage
This is the final stage in which the symptoms show no or minor cure despite seeking treatment. Syphilis symptom if left untreated it will then continue and often goes on to damage the nervous system and the heart, weakens bones & joints leading to death
None of these third stage gummas or ulcers will heal to their original state

19. Syphilis - Tertiary / Acute stage
In the central nervous system it can affect the spinal column, resulting in a condition called tabes dorsalis. This condition causes postural instability with a staggering wide base gait that can damage the joints of the leg.
The condition shown is called
Charcot's knee.
tertiary syphilis with gumma
distruction of nose

20. Syphilis - Identification of T. pallidum - Direct Microscopy
Dark field showing Treponema pallidum: A dark field analysis of fluid from a lesion of primary or secondary syphilis is considered the gold standard for diagnosing syphilis
Silver Stain
Spirochetes may be seen in biopsy specimens of suspicious lesions such as palmar macular rash or gummatous lesions

23. Case 1:
A 36-year-old female presented with a chief complaint of rash all over the body, of 15 days duration, associated with severe itching. On detailed history, the patient stated that the rash started asymptomatic on the right forearm, and later became generalized. The patient applied some native medications and developed generalized itching and presented with a picture of contact irritant dermatitis in the form of papules, vesicles, and scaling, all over the body. The patient was treated for the same with a combination of topical steroids and antihistamines. The patient had little symptomatic relief with this. The patient's rash was persistent in the form of macules, papules, and scaling. A thorough examination revealed the presence of few lesions on the genitalia mainly on the labia majora which were flat topped papules and the patient also had a mucous patch over the hard palate [. Thus, secondary syphilis was strongly suspected and the patient was subjected for Venereal Disease Research Laboratory (VDRL) testing. The VDRL testing was non-reactive. As the suspicion of secondary syphilis was high

24. Briefly outline the management of this patient
As the suspicion of secondary syphilis was high, what
further evaluation would you request to exclude or
confirm syphilis?
-Request for repeat VDRL test, with higher dilution
as false negative results will occur due to. Prozone phenomenon in
the excess of antibody( high concenteration in serum)
Also request specific Treponemal antigen tests e.g. FTA-Abs , ELISA
Briefly outline the management of this patient
secondary treatment consists in two intramuscular
of penicillin injections at weekly intervals.
treatment effectiveness will be verified on the
serology at : 3, 6 , 12 month for the drop of titers.

25. Discussion
Prozone phenomenon is an immunological event, relying on an antigen-antibody interaction such as in rapid plasma reagin (RPR) or VDRL. [3] An agglutination or precipitation reaction will be positive (i.e., visible through lattice formation) when the optimal ratio of the antigen antibody yields an insoluble precipitate that is visible, thus rendering the test positive. [4] The zone of equivalence defines this optimal ratio. In the zone of the antibody excess (prozone) or antigen excess (post zone), false negative results will occur. [4] The prozone phenomenon in the setting of syphilis may become prevalent because of the current acquired immunodeficiency syndrome (AIDS) epidemic. [2] As syphilis and HIV mutually increase the chance of contracting other diseases, B-cell behavior can lead to hyper-responsiveness to antigenic stimulation, leading to excess antibody production. [5]

27. Syphilis - Treatment of syphilis
The treatment of syphilis is variable depending on the stage of the disease:
At the primary stage: the minute treatment: a single injection of penicillin 2.4 million iu intramuscularly (except against-indication) is made and the patient will be cured once the canker is gone, it will not secondary syphilis or tertiary.
Stage secondary treatment consists in two 2.4 million iu intramuscular injections at weekly intervals. Syphilis will also be cured, treatment effectiveness will be verified on the serology.
In the tertiary stage, the treatment is longer. The effectiveness of treatment is monitored on the evolution of HIV

28. Case 2
A 35-year-old Pilipino married male presented to the emergency room complaining of dysuria for the last 24-hour and noted some "pus-like" drainage in his underwear and the tip of his penis.

29. GONORRHEA
Urethral Discharge in Gonorrhea)

30. GONORRHEA – Ophthalmia : 2-7 days after birth with
Bloody, green or serosanguineous discharge

31. What are the possible causes for his presentation?
Organisms
Urethritis
Gonococcal Urethritis
Neisseria gonorrhoeae
Purulent discharge
Non-gonococcal urethritis
Chlamydia trachomatis
Mucopurulent
Others
Trichomonas vaginatis
Mycoplasma
genitalium

32. What investigations do you like to order for him
What investigations do you like to order for him? Explain how those investigations would help you?
Organisms
Smear/Culture
Immunological tests
Molecular testing
GCU
Neisseria gonorrhoeae
Gram-ve diplococci & cellpus /
Selective media
+ve
NGCU
Chlamydia trachomatis
Pus cell/McCoy Cell culture
DFA
Others
Trichomonas vaginalis
Mycoplasma genitalium
Wet mount;
pus &TV/
Culture
Pus cell /
Special media culture
EIA

33. This Gram-stained specimen from uretheral discharge reveals N
This Gram-stained specimen from uretheral discharge reveals N. gonorrhoeae intracellular diplococci, leading to a positive diagnosis of gonorrhea

35. Base on the finding, what is the most likely diagnosis
Base on the finding, what is the most likely diagnosis? Briefly outline the management of this patient?

36. GONORRHEA
Symptoms
In both genders, gonorrhea spreads through the bloodstream to other organs, causing infection and inflammation of the joints (gonococcal arthritis), the heart (gonococcal endocarditis), or covering the brain (gonococcal meningitis).
Some adults get eye infections by rubbing eyes after touching infected genitals

37. GONORRHEA – Laboratory Diagnosis
Neisseria gonorrhoeae growing on heated blood agar (Chocolate Agar)
G.C. Sel Agar with L.C.A.T
Neisseria gonorrhoeae requires a rich growth medium, such as heated blood agar (Chocolate Agar) and an atmosphere containing 5-10% carbon dioxide. Even so, it may take up to 48 hours to get colonies of the size seen above.
Enriched and selective; contains antibiotics colistin (kills gram-negative coliforms), vancomycin (kills gram-positives), nystatin (kills fungi

38. Case 3
A 24-year-old female noted vaginal itching and irritation with a discharge. Previously, she developed a yeast infection that was treated with over-the-counter medications and resolved. Thinking that this was recurrence, she again self-treated. This time, however, the symptoms did not resolve.

39. What are the possible causes for her presentation
Bacterial vaginosis
Candida vaginitis
Trichomonas vaginalis
Allergic vaginitis
Chlamydia trachomatis
Neisseria gonorrhoeae

40. What investigations would you like to order for her
What investigations would you like to order for her? Explain how those investigations would help you? PH
Whiff test
Gram stain /
Wet prep
Culture
Immunologic/ molecular test
Bacterial vaginosis
Candida vaginitis
Trichomonas vaginatis

41. Chlamydia
Chlamydia: is currently one of the most common and widespread bacterial STDs
Spread During unprotected sex.
Symptoms: Occur within 7-21 days of infection.
Men: Clear thick discharge from Penis, burning during urination
Women: Painful Urination, itching, burning or bleeding from vagina may have vaginal discharge, dysuria, urination, labial pain/swelling, abd. Pain

42. Chlamydia Potential to transmit to newborn during delivery
Chlamydia: is currently one of the most common and widespread bacterial STDs
Spread During unprotected sex.
Responsible for causing cervicitis, urethritis, proctitis, lymphogranuloma venereum, and pelvic inflammatory disease
Potential to transmit to newborn during delivery
Conjunctivitis, pneumonia
Symptoms: Occur within 7-21 days of infection.
Men: Clear thin discharge from Penis, burning during urination
Women: Painful Urination, itching, burning or bleeding from vagina.

43. Chlamydia - Laboratory Diagnosis
Tissue culture has been the standard
Non-amplified tests
Enzyme Immunoassay (EIA), e.g.Chlamydiazyme
Nucleic Acid Hybridization (NA Probe), e.g. Gen-Probe Pace-2
detects chlamydial ribosomal RNA
able to detect gonorrhea and chlamydia from one swab
DNA amplification assays
polymerase chain reaction (PCR)
ligase chain reaction (LCR)
LCR ability to detect chlamydia in first void urine

44. What investigations you like to order for her
What investigations you like to order for her? Explain how those investigations would help you? PH
Whiff test
Gram stain /
Wet prep
Culture
Immunologic/ molecular test
Bacterial vaginosis
>4.5
+++
Clue cells
Not helpful
DNA Probe
(gardnerella vaginalis)
Candida vaginitis
<4.5 -
Yeast and pseudohyphae
Candida
Trichomonas vaginalis +-
Trichomonas
Motile Trophozoites
EIA

45. Chlamydia - Laboratory Diagnosis
2- Culture This McCoy cell monolayer micrograph reveals a number of intracellular
C. trachomatis inclusion bodies
1- Chlamydia Direct Fluorescent Antibody (DFA)
Elementery bodies
inclusion bodies

46. Chlamydia - Laboratory Diagnosis

47. CHANCROID
A sexually transmitted disease caused by a bacterium Haemophilus ducreyi)
characterized by a genital ulcer (sometimes called a soft chancre) that begins as a tender pimple surrounded by a reddened area.
It is more commonly seen in men than women, particularly uncircumcised males . Chancroid is spread by having unprotected sex.
Some symptoms include:
sores or raised bumps on the genitals which eventually if untreated, becomes filled with pus and eventually ruptures leaving a painful sore/ulcer.
The bacteria may also infect the lymph glands in the groin. These glands may become enlarged, hard and painful.
In Women, ulcers appear on the genitals, painful urination and painful intercourse
Chancroids are contagious as long as the person as open sores. 2 to 3

48. Following is the Gram-stained smear of from genital ulcer of a 25 –year old male
The organism MOST likely causing the ulcers is ; 1. Treponema pallidum 2. Herpes Simplex Virus 2 3. Haemophilus ducreyi 4. Gonorrhea

49. Chancroid ulcer Regional adenopathy
CDC / Center for Disease Control and Prevention/ Division of STD Prevention
Regional adenopathy

50. Chancroid
Diagnosis:
No blood test is available to diagnose chancroid, so the diagnosis is usually made on clinical grounds after a physical exam.

51. She presented to her family physician for management
She presented to her family physician for management. On examination there is a bad odor along with a frothy discharge and strawberry cervix.
Swab of the secretions was taken in order to perform
tests.
“Strawberry cervix”
Source: Claire E. Stevens/Seattle STD/HIV Prevention Training Center at the University of Washington

52. Wet prep/ Gram stain
A wet mount of the swab demonstrates "swimming" Motile Trophozoites .

53. Base on the finding, what is the most likely diagnosis
Base on the finding, what is the most likely diagnosis? Briefly outline the management this case?
Dx;
Trichomoniasis
Rx;
Metronidazole
Husband should be treated
No sex until they are cured
Patient Counseling and Education

54. Pubic Lice or Crabs STDs- Ectoparasitic Infestations
Lice in Pubic Area

55. Scabies STDs- Ectoparasitic Infestations Scabies causing eczema-like
hand condition
Scabies (under the skin crabs)

57. How do you diagnose HIV infection ?

58. Serological profile of HIV infection

59. DIAGNOSIS Serological test: - Screening assay (routine ELISA/rapid)
- Detection Ag or Ab
- Confirmatory assay
(Western blot, RIBA, LIA)
Direct Methods:
- PCR
- NA detection
(early inf ,neoate inf, monitor Rx )

60. Genital herpes

61. Neonatal herpes infection

62. 1- ELISA: serum sample is analyzed for detection the IgM Ab.
2- Direct immunofluorescence (IF):
scraping of the base lesion sample is analyzed for detection the Ag.
3- Polymerase chain reaction (PCR):
CSF sample in case of neonatal herpes.

63. 4- Tissue culture:
vesicle fluid sample is cultured in cell line (Vero or Hep-2 cells) and then identified by the following:
- Observe the viral CPE
- Direct immunofluorescence (IF)

64. Genital warts Diagnosis
External genital warts can be easily diagnosed by medical examination.
Internal genital warts can be visualized by colposcopy.

65. Lab diagnosis:
1- Polymerase chain reaction (PCR) is used to detect HPV DNA.
2- Pap-smear test is used to identify abnormal epithelial cells of the cervix (cervical dysplasia).
3- In-situ DNA hybridization is used for HPV genotyping.

66. THE END