This lecture was presented to the physicians dealing with the various infectious diseases specially in STIs in Riyadh Region, MOH. The lecture concentrates about the various methodology applied to diagnose STIs in the laboratory with the advantages and disadvantages of each. Hope to make benefits to all.

1. Laboratory Detection of
Sexually Transmitted Infections
(STIs)
Dr Mostafa Mahmoud, MD, Ph D,
Consultant Microbiologist
Labs & Blood Banks Admin, Riyadh.
Assist. Prof. of Medical Microbiology &
Immunology

2. What is the sexually transmitted
infections (STIs)?
 These are infections that are transmitted and
spread though the close sexual contacts.
 Having severe consequences in most cases
with many complications and even threatening
life of patients.
 Not easy to diagnose in most of infections due
to lack of symptoms or social embarrassment to
present.

3. Methods of spread:
1- Sexual intercourse.
2- Oral genital contact in sexual or in non-
sexual contact.
3- IV Drug users.
4- Congenital (Vertical or Transplacental)
Transmission from mother to baby.

4. Sexually Transmitted Diseases by organism:
Type Microorganism Infection produced
Bacterial Chlamydia trachomatis (D-K) Genital Chlamydial Infection
Chlamydia trachomatis (L1-L3) Lymphogranuloma venerum
Neisseria gonorrhoea Gonorrhoea
Treponema pallidium Syphilis
Haemophilus ducreyi Chancroid (Soft sore)
Klebsiella granulomatis Granuloma inguinale
Klebsiella granulomatis Granuloma Inguinale
Gardenerella vaginalis (!?) Bacterial vaginosis (!?)
Viral Herpes simplex I , II Genital Herpes
Papillomaviruses Ano-Genital Warts
HIV AIDS
Hepatitis Hepatitis A, B & C infection.
Protozoa Trichomonas vaginalis Trichomoniasis
Insects Pediculosis Pubis-Phthirus pubis Pediculosis

5. Presentations of Sexually Transmitted
Diseases by symptoms:
Presentation Disease or Infection
Discharge Gonorrhea
Chlamydia infections
Non-gonococcal urethritis / mucopurulent cervicitis
Trichomonas vaginitis / urethritis
Candidiasis
Sores or ulcers Chancroid (Painful)
Genital herpes (Painful)
Syphilis (Painless)
Granuloma inguinale (Painless)
Lymphogranuloma venereum (Painless)
Swellings & Tumors Warts
Cancer cervix

6. Genital Chlamydia Infection
 Transmission by unprotected sex or genital contact (not
casual contact).
 Pregnant women can pass it on to infants during birth.
 An infected person frequently has no symptoms and may
pass on infection to another.
 Up to 50% of men and 70% of women are asymptomatic
initially.

7. Chlamydia species and disease caused:
Species Serotype
(Serovar)
Natural Host Human Disease
Chlamydia trachomatis A, B, C Humana (Eye) Trachoma
(3 Biovars by type of
disease caused and tissue
tropism; trachoma,
genital infections & LGV)
with many Serovars &
genotypes.
D - K Humans (Urogenital)
Crvicitis - Urethritis
Proctitis
Conjunctivitis
Neonatal pneumonia
L1, L2 & L3 Humans (lymphoid tissue)
Lymphogranuloma
venereum
Chlamydia psittaci Birds and non-
human mammals
Pneumonia
Chlamydia penumoniae 1 Humans Acute respiratory
disease
Chlamydia pecorum Cattle and sheep ?

8. Character and pathogenesis:
 Obligate Intracellular non-motile bacteria.
 Have both RNA and DNA, ribosomes, cell wall
and divide by binary fission.
 No peptidoglycan, no ATP production.
 No growth on artificial bacterial culture media.
 Exists in 2 forms:
Elementary Body (EB): small extracellular,
infectious, metabolically inert.
Reticulate Body (RB): large, non-infectious,
metabolically active.

9. Life cycle of Chlamydia

10. Genital Chlamydia Infections:
 Caused by Chlamydia trachomatis. 106 millions
new case in 2008 globally by WHO report (The
most common STIs??).
 Incubation period: 1-3 weeks.
 Initially Asymptomatic in up to 50% of men
and 90% of women.
 Symptoms in women: include vaginal
discharge, bleeding between periods, dysuria
and lower abdominal pain.

11.  Symptoms in men: discharge from penis,
burning and itching in the genital area,
dysuria.
 Pass to neonate during birth causing inclusion
conjunctivitis (IC) and neonatal pneumonia.
 If untreated can cause serious complications in
women (PID, ectopic pregnancy, & Infertility).
 Less complications in men (epididymitis, &
prostatitis).
 Arthritis in both sexes.

12. Clinical manifestations of infection with C.
trachomatis (WHO 2013):

13. Lab diagnosis of Genital Chlamydia Infection:
 Specimens: (for culture: male urethral swab,
female endocervical swab), urine, and first void
swab from discharge.
 Lab tests: 1- the test of choice is nucleic acid
amplification techniques (NAAT) either by ligase
chain reaction (LCR) or polymerase chain
reaction (PCR). Special equipment need special
swabs supplied by manufacturers.
 Many commercial systems available for NAATs
e.g. m200 system (Abbott), BD PropeTect ET,
APTIMA Combo 2 assay (Gen-Probe, USA),
Cobas Amplicor CT/NG assay,

14. 2- Culture on cell culture rarely performed due hard
maintenance of the organism. Remember: Chlamydia
do not grow on artificial bacterial media.
- Swabs need transport media not dry one.
3- Serology: low specificity and sensitivity, for direct
detection of the Chlamydial antigens by direct
immunofluorescence assays (DFAs) or ELISA for
differentiation between chronic and acute infections.
4- Point-of-care tests (POCT) (Rapid Tests) based on
ELISA despite of being rapid have low sensitivity.
N.B. Available tests in our Riyadh MOH Labs (RRL only) are
(Chlamydia trachomatis IgG (test code 13046) IgM (code 13047),
Chlamydia pneumniae IgG (Code 13043) IgM (Code 13044).

15. Urethral swabs:

16. Endocervical swabbing

17. Evaluation of various chlamydia diagnostic tests
(WHO 2013):

19. Gonorrhea
 Caused by Neisseria gonorrhoeae (gonococcus) a
Strict human pathogen.
 Globally 106 million new cases among adults in
2008 by WHO.
 Limited to the columnar and transitional
epithelium.
 IP: 2-7 days.
 In males: presents as acute purulent discharge
from the urethra with dysuria. Can be easily
detected by Gram stain. Very rare to be
asymptomatic.

20.  In females: infection followed by
mucopurluent cervicitis which is often
asymptomatic, or vaginal bleeding after
intercourse or vaginal discharge.
 In 20% -endometritis, salpingitis with PID and
subsequent infertility and ectopic pregnancy
 Pharyngeal and anorectal lesions are usually
asympyomatic.
 Passage to neonate during birth causing
conjunctivitis (ophthalmia neonatorum) in and
may cause blindness if not rapidly and
adequately treated.

21.  In both males and females:
 Septicaemia in 0.5-1.0% of cases, endocariditis,
arthritis, skin lesions, meningitis.
 Arthritis usually polyarticular and may cause
permanent damage.
 The organism is a Gram-negative cocci mostly
intracellular.

22. Lab diagnosis of Neisseria gonorrhea infections;
 Difficulty: Gonorrhoea is frequently
asymptomatic, especially in women, and in the
pharynx, and rectum, and symptoms, if present,
can be non-specific.
 Specimens: the primacy one is endocervical
swab, Urethral Swab, Rectal swab,
oropharyngeal swab.
 The organism dies rapidly as it is highly
susceptible to environmental conditions
(temperature, desiccation, oxidation, and toxic
substances) so Dacron swab must be on
transport media (Charcoal or Amies).

23. Special swabs for NAAT by manufacturers.
Maximum time for sample storage is 48
hours stored in the refrigerator at 2–8 °C
before transportation to the laboratory.
Endocervix: Insert a swab 2–3 cm into the
cervical os and rotate gently for 5–10
seconds. Endocervical samples should not
be taken in prepubertal girls or women who
have had a hysterectomy; instead, specimens
should be sampled from the vestibule of the
vagina and a urine specimen (for NAAT
diagnostics) should also be sampled.

24.  Urethra: Take urethral specimens at least 1
hour after the patient has urinated. Collect
discharge directly on a swab. If no discharge is
evident, in men the urethra is stripped towards
the orifice to evacuate exudate. If no exudate is
obtained, insert a thin swab 2–3 cm into the
urethra and gently rotate for 5–10 seconds.
 In women, massage the urethra against the
pubic symphysis and use the same technique as
for men. Culturing both endocervix and urethra
when testing women can increase case finding.

25.  Vagina (NAATs only): The swab should be
rotated against the posterior vaginal walls for 5
seconds.
 First void (catch) urine (NAATs only): Do
not have the patient clean the genital area.
Catch 10–20 ml of first void urine in a sterile
collection container at least 1 hour after the
patient has urinated.
 Rectum: Insert a swab 2–3 cm into the rectum
and rotate it against all the rectal walls for 10
seconds. If fecal contamination occurs, discard
the swab and use another to obtain the
specimen.

26.  Oropharynx: Swab the region of the
posterior pharynx above the inferior edge of
the soft palate and the tonsillar crypts.
 Conjunctiva: Retract the inferior eyelid and
move a thin swab across the surface of the
inferior palpebral conjunctiva towards the
median corner of the eye.

27. Lab Testing for Neisseria gonorrhea infections;
1- Direct Gram-stain: showing intracellular
diplococci in polymorphonuclear leukocytes
(PMNL) is sensitive (95%) and specific (97%)
for the diagnosis of gonorrhoea in
symptomatic men with urethral discharge.
 In women, only positive in 40-60% of
cervical secretions.

28. Direct Gram stain of Neisseria gonorrhea

29. 2- Culture: was the gold standard for diagnosis
before NAAT test. Needs special media so hint
to the diagnosis must be written in the request
(Chocolate agar, Thayer-Martin, New York City,
Muller-Hinton agar) with 5-10% CO2 during
incubation.
 Culture is very important to perform
antimicrobial susceptibility testing (AST) due
to presence of resistance nowadays.
 N.B. Available tests in our Riyadh MOH Labs are Direct Gram stain, and
Culture (in all Hospitals with microbiology Lab). No serology no NAAT no
POCT.

30. Candle jar for 5-10% CO2

31. CO2 Incubators.
??

32. Growth of Neisseria gonorrheae
On chocolate agarOn Thayer-Martin agar

33. 3- NAAT assays for detection of DNA or RNA of
Neisseria gonorrhea were developed lately and
are more sensitive than culture especially for
rectal and pharyngeal infections.
 Serology :up –to-date, not available either ELISA
or DFAs for direct antigen detection . Also no
available test for antibody detection in serum to
differentiate acute from chronic infections..
 Rapid point-of-care tests for antigen detection
not available with appropriate performance
characteristics (sensitivity and specificity).

34. Common diagnostic tests for detection of N.
gonorrhoeae WHO 2013:

35. Syphilis
 Syphilis is a chronic sexually transmitted disease
characterized by many clinical manifestations
and long periods of quiescence.
 It is caused by Treponema pallidum subsp.
pallidum, which is a venereal spirochaete.
 Non-venereal treponematoses, include T.
pallidum subsp. pertenue (yaws), T. pallidum
subsp. endemicum (endemic syphilis), and T.
carateum (pinta).
 Sexually transmitted and transplacenatlly
(congenital infection).

36.  Systemic infection and the organism enter blood
stream.
 T. pallidum cannot be cultivated on artificial
media only preserved on rabbit testicle.

37. Stages of non-treated syphilis:

38. Lab diagnosis of syphilis:
 I- Direct detection methods:
A- Dark-field microscopy is the only point-of-
care (POC) method that is capable of
establishing a direct diagnosis of syphilis in
cases of adult primary or secondary or early
congenital disease.
 Special microscope is needed.
 Needs to differentiate T. pallidum from non-
pathogenic treponemes and other spiral
organisms commonly found on genital organs.
 Specimens are serous exudate from active lesions.

39.  T. pallidum appears as bright, white spiral
bodies, motile like spring, illuminated against a
black background.
 Causes of false negative results: low number of
organisms, Antimicrobial treatment, natural
resolution of the stage.

40. B- Direct fluorescent antibody (DFA) test:
 Specimen collection same like dark-field.
 Smear on slide, air-dry, fix with acetone or
methanol, treat with fluorescein-labelled anti-
T. pallidum globulin, wash excess then examine
by Fluorescent microscope.
 The organism appears apple-green in color.

41. C- Nucleic acid amplification tests for T.
pallidum:
 Performed by PCR technology.
 Has the advantages of testing any specimens from
any lesion exudate, tissue, or body fluid, and the
specimen can be fresh, frozen, or fixed and
paraffin-embedded.
 Can detect down to 10 organisms so positive in
neurosyphilis and congenital syphilis and
organisms in blood.
 Multiplex PCR assays have been developed for the
simultaneous detection of the most common
causes of sexually transmitted genital ulcer disease
(GUD), namely T. pallidum, Haemophilus ducreyi,
and herpes simplex virus.

42. II- Serological testing for syphilis
 Standard Tests for Syphilis (STS) two categories:
A- Non-treponemal serological tests:
Wasserman reaction (WR), rapid plasma reagin
(RPR), Venereal Disease Research Laboratory
(VDRL), and toluidine red unheated serum test
(TRUST) tests.
B- Treponemal Tests: FTA-Abs, T. pallidum
haemagglutination assay (TPHA), T. pallidum
passive particle agglutination assay (TPPA),
ELISA, chemiluminescence, and the vast majority
of POC or rapid tests. Use Specific Ag.

43. A- Non-treponemal serological tests:
 Detect non-specific Antibodies (Reagin) (IgG & IgM)
reacting with non-specific Ag (mixture of cardiolipin,
lecithin, and cholesterol).
 Rapid tests but lacking specificity with 0.2-0.8% of test
with false positive in malaria, hepatitis, chicken pox, or
measles, or with recent vaccination.
 Also false +ve in connective tissue disorders,
malignancies, chronic infections such as leprosy,
intravenous drug abuse, and ageing and may be
pregnancy.
 Better to be used for screening to be confirmed.
 The early test was WR based upon complement
fixation test (CFT) rarely used nowadays.

44. 1- Venereal Disease Research Laboratory
(VDRL) test
 Antigen used in test is not stable need fresh
daily preparation.
 Test on heated serum 56 OC.
 Performed over microscopic slides.
 Test needs microscope to read flocculation.
 Not used nowadays but of choice in CSF
sample.
 Can be used qualitatively or quantitatively.

45. VDRL Slides in India

46. 2- Rapid Plasma Reagin (RPR) test
 Antigen used in the test is stable and can be
kept long shelf life.
 Cards instead of slides.
 Flocculation seen by use of charcoal particles.
 No heating of serum.
 No microscope for reading results (Naked eyes).
 The test currently used for screening in our
(MOH) laboratories.
 Has same limitation of non-treponemal tests.

47. RPR Test

48. Reactivity of non-treponemal serological tests during the course
of untreated syphilis (boxes) and response of tests following
successful therapy (circles), by stage of disease (WHO 2013)

49. B- Treponemal Serological Tests:
 More specific.
 Very rare false positive result with connective
tissue disorders.
 No preferred to monitor response to syphilis
therapy as most of them become positive for life
and better to use the non-treponemal serological
test for that purpose.

50. 1- The Fluorescent Treponemal
Antibody Absorption (FTA-Abs) test
 Was the specific test of choice but now there
more specific tests.
 Needs fluorescent microscope.
 Use specific Antigens to detect
specific Antibodies.
 Use Fluorescein isothiocyanate (FITC)-
labelled antihuman immunoglobulin
conjugate.
 False positive due to subjective error in
reading the test.

51. 2- Treponemal enzyme immunoassays
(EIAs) and chemiluminescence assays
(CIAs).
 Recent test with near sensitivity and specificity
to FTA-Abs and agglutination assays.
 Use sonicatd or recombinant Treponema
Antigens to detect specific antibodies in
patient serum.
 Automation is available. (Used in MOH)
 Many manufacturers and kits are available.

53. 3- Treponemal western blot (WB) assays:
 Confirmatory test to detect specific IgG & IgM
Antibodies in patient serum.
 Test is not in common use now.
4- Line immunoassays
5- POC syphilis tests
• Many different test and kits are available.

54. Detection of antibodies to Treponema pallidum by western
blot (WB)

55. POCT Rapid test for syphilis:

56. POCT Rapid test for syphilis:

57. Reactivity of treponemal serological tests during the course of
untreated syphilis (boxes) and response of tests following
successful therapy (circles), by stage of disease (WHO 2013).

58. Performance characteristics of selected
serological tests for syphilis by stage of disease

59. Other rare diagnostic tests for syphilis
1. Rabbit infertility test (RIT). High specificity,
delayed result, for Research only
2. Treponema Pallidum immobilization (TPI)
test (Highly dangerous test using living
organism).
3. Treponema pallidum haemagglutination
test- TPHA (available in our MOH)
4. Biopsy to show endarteritis obliterans with
inflammatory reaction or granuloma in 3ry
syphilis.

60. The laboratory diagnosis of congenital syphilis
 Skin lesions: dark-field microscopy, direct IF, or
PCR for direct evidence of infection with T.
pallidum.
 Serology is not diagnostic due to passive transfer
of antibodies, but > four-fold increase of
RPR/VDRL than mother titre is diagnostic.
 FTA-IgM Abs test (FTA-IgM) as IgM cannot cross
the placenta.
 IgM-specific EIAs

61. Neurosyphilis diagnosis:
 Lumbar puncture: VDRL-CSF test performed, high
specificity (99–100%) but low sensitivity for
neurosyphilis.
 FTA-Abs CSF test has high sensitivity but low
specificity (passive transfer of IgG to blood brain
barriers).
 N.B. Available tests for syphilis in our Riyadh MOH
labs are RPR (All Labs), TPHA (Most labs with
microbiology & RRL).

62. Diagnostic Algorithm for Syphilis
Non-treponemal test Screening (e.g. RPR)
Treponemal test (e.g. FTA, EIA) Negative for syphilis
Non-reactive
Non-reactive
Syphilis
Negative for syphilis
Reactive
Reactive
25 62

63. Chancroid (soft sore)
 Painful genital ulcer (penile coronal sulcus in
men and the vulva in women) and tender
inguinal lymph adenopathy.
 The causative organism is Hemophilus ducreyi
invading intact skin or mucous membranes.
 Gram-negative coccobacilli in short chains,
clumps, or whorls.
 IP: 4-10 days.

64. Lab diagnosis of Chancroid:
 Specimen for diagnosis to be taken from the base
of the ulcer after cleaning with gauze or dry
swab. Use swab with transport media kept at 4
oC.
1- Direct microscopy has very low sensitivity and
specificity (Not used)
2- Culture to recover H. ducreyi (Gold standard).
Need special media for inoculation at 5% CO2 at
32–34°C for 48 hours up to 5 days incubation is
needed. (Available in all MOH Microbiology Labs).
3- AST needed for the selection of antimicrobial.

65. 4- Antigen detection of H. ducreyi for research
only not for diagnosis.
5- Serology: are not currently commercially
available.
6- Nucleic acid-based detection of H. ducreyi not
available.

66. Granuloma Inguinale (Donovanosis)
 It is a genital ulcerative disease caused by the
Gram-negative, intracellular bacterium
Klebsiella granulomatis (formerly known as
Calymmatobacterium granulomatis).
 It is painless very vascular ulcer with no
lymphadenopathy.
 It is endemic in some tropical and developing
areas, including India; Papua, New Guinea; the
Caribbean; central Australia; and southern
Africa. Very rare in USA.

67. Lab diagnosis:
1- Very difficult to culture
2- Direct microscopy using dark stains is the
diagnostic tool. (Available in all Riyadh
Region MOH Microbiology Labs).
3- No NAAT available,

68. Bacterial Vaginitis or vaginosis
 The most common cause of vaginal discharge in
childbearing age.
 Organism of Controversy: STD - yes or no??.
 Not sexually transmitted by sex activity increase
it.
 Caused by many of the vaginal folra mainly
various anaerobes and Gardnerella vaginalis are
increased with reduction of lactobacilli.
 Associated with increased risk of many
gynecological diseases.

69. Lab diagnosis
 Combined clinical and lab test
 Fishy odour
 Positive amine test intensify odour by 10% potassium
hydroxide,
 High vaginal pH > 4.5
 “clue cells” visible on microscopic examination (Gram).
 Point of care test is available.

70. Genital Herpes Simplex
 Herpes simplex virus type 1 (HSV-1) and type 2
(HSV-2) are large double-stranded DNA viruses.
 Infection are by direct contact via break in skin
and sexual.
 Primary infection is symptomatic in 10-30% of
infections.
 There is recovery followed by latency in the
sensory ganglia for life and reactivation later on
may occur.
 Recurrent symptoms are milder than primary
infection.

71. Clinical picture of genital herpes:
Clinical symptoms and symptoms Complications
Males • Papular or vesicular lesions on genitals, perigenital
areas, or extragenital areas (thigh, eye, buttock,
finger)
• Pustular lesions • Genital ulceration
• Perineal pain • Dysuria
• Inguinal discomfort or pain (peri-adenitis)
• Pharyngeal infection (oro-genital contact)
Urethral discharge Urethritis
• Aseptic meningitis
• Extensive vesicular
skin rash
• Urinary retention
• Sensitive
radiculopathy by
involvement of sacral
nerves
• Transverse myelitis
• Increased risk for
acquiring HIV by
sexual exposure
• Increased risk of
sexual transmission
of HIV in
HIV/HSV-coinfected
individuals
• Neonatal herpes
after vaginal delivery
Females • Papular or vesicular lesions on genitals or perigenital
areas or extragenital areas (thigh, eye, buttock, finger)
• Pustular lesions
• Genital ulceration • Vaginal or cervical discharge
• Perineal pain Dysuria
• Inguinal discomfort or pain (peri-adenitis)
• Dyspareunia
• Pharyngeal infection (oro-genital contact)
• Primary infection is often worse in intensity
and duration in women than in men.
congestion of vulva and vagina • Urethral discharge
• Urethritis.

72. Lab diagnosis of genital herpes infections:
 Correlated clinical picture as a result of the presence of
a cluster of vesicular lesions but caused by many
organisms.
 Specimen: swabbing mucocutaneous genital lesions by
dry cotton-tipped or Dacron swab for NAAT. collection
of vesicular fluid or exudate by tuberculin syringe. Use
viral transport medium (VTM) for cell culture.
 Send samples to the laboratory on ice in a cooler box
freezing not recommended.
1- Direct microscopy by stained cytology.
2- detection of HSV Antigen in material from lesions.
3-Viral culture.
4- NAAT (the preferred test)
5- indirect Serology for antibody (low value).

73. 1- Direct microscopic (Cytological) examination:
- Tzanck smears, Papanicolaou (Pap), or
Romanovsky stain using Skin/mucosal
lesions, Biopsies or Conjunctival/corneal
smears of low sensitivity and specificity and
needs fresh samples but inexpensive.
- Detection of infected cells by direct
immunofluorescence (IF) using smears,
tissue of moderate to high sensitivity and
specificity inexpensive, time-consuming, not
standardized.

74. 2- Viral antigen detection:
i- immune-peroxidase (IP) or
immunofluorescent (IF) staining on swabs or
smears or vesicular fluid. Rapid but costy with
suboptimal sensitivity.
ii- ELISA Test: on swabs or smears or vesicular
fluid. High specificity and sensitivity but
needs fresh vesicles.
iii- Rapid test (POCT) devices on swabs or
smears or vesicular fluid. Unknown
specificity and sensitivity. (not yet evaluated)

75. Intracellular immunofluorescence of HSV in a
clinical specimen

76. 3- Viral culture
Specimen: • Swab, Skin lesions, Vesicular,
fluid or exudate from base of vesicle, Mucosa
sample without lesions, Biopsies,
Conjunctival/corneal smear& Neonates.
Viarible sensitivity and 100% specificity.
- Adv. Gold standard for diagnosis, simple
sampling, viral typing.
- Disadv. Sample storage & Transport affects
results, cumbersome, expensive, late results (2-7
days), less sensitive than PCR.

77. 4- Molecular assays:
- For detection of viral DNA by NAAT via PCR or
real-time PCR.
- Specimens: swabs, skin lesions, vesicular fluids or
exudates, mucosal sampling without lesions, CSF,
Blood.
- The highest specificity and sensitivity.
- Adv. Rapid, high specificity and sensitivity, allows
typing, automated, the preferred test currently.
- Disadv. Specialized lab, real-time PCR expensive,
risk of contamination (false-positive).

78. 6- Indirect serological diagnosis of herpetic
infections:
Serological testing is recommended as an aid to
diagnosis of genital herpes in patients with
recurrent genital symptoms, atypical lesions, or
with healing lesions and negative HSV cultures.
- Low sensitivity and specificity.
- For detection of antibodies IgG and IgM.
- N.B. Available tests in our Riyadh MOH Labs (RRL
only) are HSV1 IgG (test code 13052) IgM (code 13051),
HSV2 IgG (Code 13054) IgM (Code 13054) HSV 1&2 IF
(code 13122) HSV DNA 1&2 PCR (code 80027).

79. Human Papillomavirus (HPV) infections
- Small viruses with circular ds-DNA, non-
enveloped.
- Do not grow on tissue culture so can not be
serologically or antigenically typed.
- Only genotypically typed.
- Infects skin and mucosal surfaces of humans.
- Most infections are transient and cleared by host
immune system but in 5-10% persistent infections
occur.

80. - Cofactors for persistence and cancers
include: cigarette smoking, long-term oral
contraceptive use, high parity, early age at
first delivery, and immunosuppressive states
such as HIV coinfection
- They cause anogenital warts and cancers.
- Based on carcinogenicity; HPV genotypes
are low risk (LR) e.g. 6 & 11 or high risk (HR)
16 & 18.
- Vaccine available for high risk genotypes.

81. Laboratory Detection of HPV:
1- Molecular testing is the gold standard for
detection of the HR genotypes 16, 18, 31, 33, 35, 39,
45, 51, 52, 56, 58, 59, 66, 68.
-Primary screening for women > 30 years old.
-For women with minimal cytology abnormalities.
-To monitor recurrence
-Methods (Many Assays): i- Direct-probe
hybridization assays for detection of the DNA of
suspected virus genotype e.g. The Hybrid Capture
test (HC2) (Qiagen),-
ii- HPV mRNA assays: to detect mRNA expression
of E6/E7 oncogenes.

82. 2- No viral culture is available as it doses not
grow on tissue culture.
3- Serology is insensitive and used only for the
purpose of sero-surveillance.
N.B. Available tests in our Riyadh MOH labs
(RRL only) are HPV type 16/18 In-Situ
hybridization (test code 50115), HPV type
31/33/51 In-Situ hybridization (test code 50116).

83. FDA-approved HPV tests (WHO 2013)

84. HIV
HIV-Structure (Viral Antigens)
1- Gag= Group Antigen: (P24 (Capsid), P17
(Matrix), & P7 (Nucleocapsid).
2- Pol= polymerase and associated enzymes:
P66 (reverse transcriptase -RT), P32 (integrase) ,
P9 (protease).
3- Env= Envelope glycoproteins e.g. GP 120, GP 41.

86. HIV Diagrammatic Representation

87.  The effects of HIV are due to it gp 120 Ag.
 The CD4+ are the receptors of the virus.
 HIV affects the immune system & Brain??
 Why?
 They have CD4+Tmainly on T-helper cells
 Other cells having CD4+ include:
- Macrophages - Dendritic cells.
- Monocytes - Microglial cells
- Retinal cells - Colonic Mucosal
cells
HIV infects (CD4+) carrying cells!!!

89. A- Tests to diagnose HIV infections
1- Screening 2- Confirmatory
B- Tests for follow up the disease;
1- Viral load by Quantitative PCR
2- Th-cells count (CD4+)
C- Tests for the complication of the disease
1- TB infection 2- HBV
3- HCV 4- Toxoplasma
5- Liver function tests 6- UTIs
7- Others.
Diagnostic lab test for HIV infection

90. A- Tests to diagnose HIV infections
1- Screening tests
These test are rapid in giving results however,
they are not diagnostic and need confirmation by
the confirmatory tests
The Screening tests include:
i- ELISA tests (combined Ag-Ab Immunoassays)
for detecting HIV-1 & HIV-2 antibodies and P24
Ag of HIV-1. or chemiluminescent immunoassay
testing.
ii- Rapid tests

91. Indications for HIV screening tests:
1- Premarital examination
2- Blood Donors.
3- Antenatal Care
4- Pre-employment screening
5- Before Surgical operations (Medico-legal)
6- Follow up for hemodialysis patients.
7- Others

92. Manual ELISA readers
After several incubation and
wash steps, a color reaction
occurs if HIV antibody is
present
An automated reader gives
a measurement of optical
density (presence of color)
for each well

93. Fully Automated ELISA machines (dilution,
pipetting, washing, incubation and reading)
Evolis (Bio-Rad) Eti- Max (Diasorin)

94. ELISA KITS IN CURRENT USE

95. ii- Rapid HIV tests Advantages:
 Easy to use immediate and fast results (10-
20 minutes).
 Can be used at home or clinic.
 Inexpensive (1-2 $/test)
 Recently approved by MOH (KSA)
 Some tests are FDA approved
 Done upon whole blood or saliva
 No equipment, refrigeration, No electricity,
No multiple timing steps required.
 Built in controls

96. Disadvantages of HIV rapid tests.
 Only detects Antibodies to HIV 1/2
 Not detecting Antigens
 Positive late after infection; after
seroconversion.
 Need confirmation for reactive tests.
 Subjective variability in result reading.

97. Rapid tests for HIV diagnosis

98. Home HIV Rapid tests.

99. Clinic Rapid Tests.

101. 2- HIV Diagnostic Confirmatory tests
1- Differential Antibody tests (Western Blotting,
WB).
2- Indirect Immunofluorescene Assay (IFA). Not
available in MOH
3- Nucleic acid Amplification tests (NAT, PCR)
done in most (MOH) hospitals for
screening blood donors and in RRL for
confirmation of screening tests.
4- Virus Isolation (not done for clinical diagnosis
only in research purposes).

102. Western Blot for detection of various
antibodies to various parts of the virus

103. CDC Recommended Laboratory HIV Testing
Algorithm for Serum or Plasma Specimens
(WB)
(ELISA)
(PCR)

104. REASONS FOR FALSE-POSITIVE, FALSE-NEGATIVE, AND
INDETERMINATE RESULTS IN ASSAYS FOR THE DETECTION
OF ANTIBODIES AGAINST HIV
Reasons for
False-Positive
HIV Screening
Test Results
• Increased sensitivity of assays, leading
to reduced specificity
• Technical errors
• Presence of HIV antibodies in
recipients of HIV-1 trial vaccines.
Other rare possibilities:
• Hypergammaglobulinemia/antibodies
reactive to cellular components
• Influenza vaccination may cause cross-
reactivity with HIV antibody assays. The
time course for such cross-reactivity
remains uncertain.

105. Reasons for
False-
Negative
HIV
Screening
Test Results
• Testing individuals during the window period
(the incubation period between exposure and
seroconversion) technical errors
• HIV-2 (for tests designed to detect).
 Other rare possibilities:
• Delayed antibody synthesis in infants and
persons receiving post-exposure prophylaxis or
with concurrent acute hepatitis C infection
• Diminished immune response in individuals
receiving intensive or long-term immune-
suppressive therapy
• Congenital or drug-induced hypogamma-
globulinemia or agammaglobulinemia
• Insufficient host antibody response (i.e.,
advanced HIV disease)
• Unavailability of antibodies due to the
formation ofgen-antibody complexes
• Reduced sensitivity assays

106. Reasons
for
Indetermi
nate*
Western
Blot
Results
(positive
screening
and
negative
WB)
 Probable True Positive (HIV Infection)
• Seroconverting
• HIV-2 infection
• Technical errors
 Probable True Negative (No HIV Infection)
• Recipients of HIV-1 trial vaccine
• Antibodies reactive to cellular components, as in
o Multiparous women
o Polytransfused patients
o Patients receiving chronic hemodialysis
o Patients with autoimmune disease
• Recipients of influenza and hepatitis B virus
vaccines
• Persons with non-HIV acute viral infections
• Congenital bleeding disorders
• Alcoholic hepatitis and other chronic liver diseases
• Hematologic malignancies, lymphomas
• Positive rapid plasma reagin test

107. Other lab tests for HIV patients

108.  N.B. Available HIV tests in our Riyadh MOH Labs
are HIV Rapid tests (All PHCs) and some hospital
clinics, HIV I & II Ag-Ab combo ELISA test (All
Riyadh Hospitals), HIV Multiplex NAAT (most
hospitals with blood bank facilities), HIV 1&2
Confirmatory test (RRL, Test code 13025), HIV
RNA PCR (RRL test code 80024), HIV P24 Ag
Confirmation (Test code 13027) .

109. Hepatitis A, B, & C
 Hepatitis A virus (HAV) is transmitted faeco-
orally with evidence for sexual transmission
between homosexual men.
 Testing for HAV is done in case of outbreak or
for homosexual and contacts to unknown cases.
 Hepatitis B virus (HBV) infection is
transmitted vertically (mother to child),
parenterally and sexually
 Hepatitis C virus (HCV) is transmitted
parenterally with a low rate of sexual and
vertical transmission.

110. Lab diagnosis of Hepatitis A, B, C
 Hepatitis A (HAV):
 POCT (low sensitivity, 80%)
 ELISA for anti-HAV IgM (Sensitivities and
specificities approach 100%).
 Hepatitis B (HBV):
 POCT rapid tests.
 Screening in asymptomatic patients may
include tests for HBsAg, anti-HBc and anti-HBs
(ELISA or immunochemiluminscent)
 HBV-DNA NAATs testing by PCR

111.  Hepatitis C (HCV):
 POCT rapid tests:
 Screening by ELISA for detection HCV-Ab
 Confirmation by RIBA for HCV-Ab
 NAAT assays for HCV- RNA quantitative or
qualitative assays.
 N.B. Available tests in our Riyadh MOH Labs are HBsAg
screening ELISA (all hospitals) HCV Ab Screening ELISA (All
hospitals), HBsAg confirmation (RRL Code 13006 and some
hospitals), HBcAb (Hospitals with blood banks) other HBV
Markers by ELISA (RRL only many codes), HCV RNA PCR Quant.
(RRL Code 80022), HBV DNA PCR (RRL only code 80019), HCV
Genotype (RRL only 80020), HAV IgM ELISA (RRL only code
13015) HAV Total Ab (RRL only Code 13014).

112. Rapid tests for HBV, HIV, & HCV

113. Trichomoniasis
 It is caused by the protozoan Trichomonas
vaginalis with estimated 276.4 million new cases
globally in 2008 (> Chlamydia & Neisseria).
 The organism is motile, ovoid or pear-shaped
flagellated protozoan.
 In women it causes vaginal discharge and in men
it is mostly asymptomatic.
 Other manifestations include; female: dysuria,
pelvic pain, itching. Male: dysuria, discharge,
and testicular pain.
 Infection peak it at age of 40-50 years of age.

114. Lab diagnosis of Trichomoniasis:
1- wet preparation microscopy:
- Can be in clinics needs immediate examination
after collection (within 10 minutes).
- if positive (definitive diagnosis).
- Organism is highly sensitive to temperature
losing motility (hallmark) after 10 minutes.
- Needs high number of organism to be detected.
- Microscopy has low sensitivity (40-65% for
women).

115. TV Wet exams

116. 2- Antigen Detection (POCT):
- Many assays are available.
- Variable costs and sensitivity but higher than wet
microscopy (OSOM Trichomonas Rapid Test).
3- Culture:
- was the main diagnostic test but now no more.
- The organism can grow anaerobically at 37 oC for
7 days .
- Special media modified Diamond’s medium or
InPouch TV culture system.
- Time consuming and low sensitivity than NAATs

117. TV Ag Detection POCT rapid tests

118. InPouch TV Culture system:

119. 4- NAATs
- The highest sensitivity and specificity and great
flexibility in sample collection.
- Automation is possible.
- Rapid test results.
-Disadvantage of expensive costs and needs
specialized laboratories.
-Only direct microscopy is available in our MOH
Riyadh Region hospitals), Culture in RRL only code
01040).

120. Pediculosis Pubis-Phthirus pubis (No Lab)

121. Pediculosis

122. To avoid all these diseases it so easy to avoid
illegal unprotected sex. As our Quran saying
(do not approach unlawful sexual intercourse .
Indeed, it is ever an immorality and is an evil
as a way).

123. References: Laboratory diagnosis of sexually transmitted infections, including
human immunodeficiency virus. WHO. 2013
 Centers for Disease Control and Prevention and Association of Public
Health Laboratories. Laboratory Testing for the Diagnosis of HIV
Infection: Updated Recommendations. Available at
http://stacks.cdc.gov/view/cdc/23447. Published June 27, 2014.
Accessed [30/11/2015].
 Diagnosis of HIV-1 Infection. Estelle Piwowar-Manning, HPTN
Central Laboratory. The Johns Hopkins University.
 Sexually Transmitted Infections: UK National Screening and Testing
Guidelines . 2006.
ABBREVIATIONS:
 MOH= ministry of health , RRL= Riyadh Regional Lab.

124. Thank you