Bacterial Sexually Transmitted Infections

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Bacterial Sexually Transmitted Infections

• Patrick Kimmitt

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Today we are going to look at

• Three distinct bacterial pathogens causing
  sexually transmitted infections
• Neisseria gonorrhoeae
• Chlamydia trachomatis
• Treponema pallidum

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We are going to consider

• The organism, structure and physiology
• The pathology of disease
• Epidemiology
• Laboratory diagnosis and treatment
• There are many contrasts when looking at these
  uniquely adapted pathogens
• You should be able to discuss each of these
  aspects

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Gonorrhoea

• Neisseria gonorrhoeae

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Clinical and epidemiological aspects

• 2nd commonest bacterial STI
• 2007 18,710 cases reported to HPA
• Most common age groups males 20-24
  females 16-19
• Males usually symptomatic
• Females often asymptomatic
• Complications untreated females PID,
  infertility, ectopic pregnancy

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Number of diagnoses of gonorrhoea by sex, GUM
clinics, England and Wales 1925 2006
Scotland Northern Ireland data are excluded
as they are incomplete from 1925 - 2003 Routine
GUM clinic returns
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Rates of diagnosis of uncomplicated gonorrhoea by
sex and age group, GUM clinics, United Kingdom
1997 - 2006
Females
Males
Routine GUM clinic returns
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Number of diagnoses of uncomplicated gonorrhoea
by sex and male sexual orientation, GUM clinics,
United Kingdom 1997 - 2006
Routine GUM clinic returns
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Symptoms (if present)

• Males urethral discharge, severe burning on
  urination
• Females vaginal discharge, yellow or
  blood-stained, pain on urination
• Rectal infection gives rise to pain and discharge
• Pharyngeal infection, sore throat

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Symptoms 2

• Both sexes disseminated infection on rare
  occasions usually as septic arthritis
• Infection during pregnancy may lead to ophthalmia
  neonatorum of baby (conjunctivitis)- blindness
• May see dual genital infection with Chlamydia
  trachomatis usual to treat for both at time of
  gonorrhoea diagnosis

For more info Kimmitt et al Journal of Travel
Medicine (2008) 15 369-371
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Neisseria gonorrhoeae

• The causative organism is Neisseria gonorrhoeae,
  a Gram-negative diplococcus i.e. often see cells
  as a pair. The genus Neisseria contains one
  other pathogenic species, N. meningitidis, which
  is the principle cause of bacterial meningitis.
  There are also many non-pathogenic species of
  Neisseria, often found in the pharynx

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Gram stain from a clinical sample
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Neisseria gonorrhoeae

• N. gonorrhoeae is phagocytosed by
  polymorphonuclear neutrophils but resists
  intracellular destruction, remaining intact
  within the neutrophil.
• It is fastidious, sensitive to desiccation and
  requires aerobic incubation with 5 carbon
  dioxide for growth. It grows as a small colony,
  often requiring 48 hours incubation. The
  colonies are grey, shiny, often with an irregular
  edge. The organism is catalase positive and
  rapidly oxidase positive.
• No protective antibody response to gonorrhoea
  recurrent infections are common in people who are
  at risk.

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Laboratory methods

• Culture is required - for identification and
  antibiotic sensitivity tests
• Urethral, cervical, rectal or pharyngeal swab
• Use selective medium containing antibiotics and
  growth supplements (look this up)
• e.g. Thayer Martin or New York City media
• Molecular tests have been developed for the
  direct detection of N. gonorrhoeae infection and
  a single swab may be used in a double test to
  detect N. gonorrhoeae and Chlamydia trachomatis.
• Commercial tests include the COBAS Amplicor and
  SDA tests

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Identification tests

• Once you have cultured your samples you need to
  perform tests on single colonies to check/confirm
  identification
• Oxidase test - result?
• Gram stain what are you looking for?
• Phadebact GC uses a specific monoclonal
  antibody
• API NH utilizes carbohydrates plus enzymes
  activity, similar to API 20E

N. gonorrhoeae is often referred to as a
gonococcus or GC
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Treatment

• There is increasing resistance to penicillin and
  now ciprofloxacin
• The recommended treatment of gonorrhoea is now
  either ceftriaxone (injectable) or cefixime
  (oral). As yet, no resistance has been reported
  to these third generation cephalosporins. In
  either case, a single dose is all that is
  necessary for the treatment of non-disseminated
  gonorrhoea

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Chlamydia

• Chlamydia trachomatis

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Clinical and epidemiological aspects

• The most common bacterial sexually transmitted
  infection, with 121,986 cases reported to the
  Health Protection Agency in 2007
• The causative organism is Chlamydia trachomatis
• The number of cases has risen steadily since the
  mid 1990s

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Rates of diagnoses of uncomplicated genital
chlamydial infection by sex and country, GUM
clinics, United Kingdom 1997 - 2006
Males
Females
Routine GUM clinic returns
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Rates of diagnoses of uncomplicated genital
chlamydial infection by sex and age group, GUM
clinics, United Kingdom 1997 - 2006
Males
Females
Routine GUM clinic returns
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Chlamydia STIs

• We have known about Chlamydia causing STIs for
  many years but it is only in the last 10-15 years
  where we have seen it emerge as a major pathogen
• Most common age groups males 20-24
• 
  females 16-19
• Government screening for Chlamydia in under 25s
  announced in 2003

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National Chlamydia Screening Programme

• NCSP aimed to screen at least 15 of sexually
  active 16-24 year olds.
• 70m invested aim to reduce the burden of
  disease due to Chlamydia
• Hospital labs have seen a dramatic increase in
  their Chlamydia testing workload
• Is it working?

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Chlamydial disease

• Also known as NSU or NGU
• The infection has a longer incubation period than
  gonorrhoea, of 1 to 3 weeks compared to 2-3 days
  (usually)
• As symptoms for gonorrhoea appear first this is
  why treatment for both infection is usually
  offered
• Asymptomatic Chlamydial infection is common in
  both sexes at least 50 in males and 70 in
  females

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Symptoms (when present)

• Females
• unusual vaginal discharge
• bleeding (intramenstrual)
• pain on urination
• lower abdominal pain
• Males
• urethral discharge
• burning and itching in genital area
• pain on urination

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Symptoms

• In some cases the symptoms subside after a few
  days
• In either sex, complications may ensue in the
  case of untreated infection
• In males, untreated infection may lead to
  epididymitis and Reiters Syndrome (arthritis)
• In females, the consequences of untreated
  infection are pelvic inflammatory disease (PID)
  in 10 to 40 of cases

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Symptoms

• In up to 20 of patients with PID, infertility
  develops and the risk of ectopic pregnancy
  increases
• The risk of infertility also increases if there
  has been more than one episode of PID
• Infection in pregnancy can lead to infection of
  the baby - trachoma inclusion conjunctivitis or
  pneumonia

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Chlamydia lifecycle

• Chlamydia is an unusual bacterial genus it is
  an obligate intracellular pathogen
• Over time it has lost the capacity to replicate
  independently
• How would this affect laboratory diagnosis?

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Lifecycle

• During their lifecycle Chlamydia may be found in
  two forms elementary bodies and reticulate
  bodies
• the infective form of Chlamydia is the Elementary
  Body (EB), a dense, circular body, about 0.3µm in
  diameter. EBs are fairly inert and can survive
  outside the cell

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Life cycle

• EBs carry glycosaminoglycan molecules on their
  surfaces that bind to receptors on the surface of
  certain cells
• after attachment, the EB is taken into the cell
  by endocytosis and remains inside the endocytotic
  vacuole for the next phase of the life cycle

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Life cycle

• the EB develops into a Reticulate Body (RB) which
  is larger (0.5 to 1.0µm) and metabolically
  active, although it uses host cell ATP-generating
  systems
• inside the vacuole, the RB grows and replicates
  its DNA
• during this phase, the contents of the vacuole
  are termed an Inclusion Body

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Life cycle

• Staining of the Inclusion Body with iodine
  todemonstrateinfection of cellcultures

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Life cycle

• EB Formation and Release
• after 18 to 24 hours,the RB reorganisesinto
  many EBswhich are releasedon cell rupture(24
  to 48 hoursafter infection)

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Chlamydia trachomatis

• There are many different serotypes and these can
  be grouped according to the type of disease that
  they cause not all infections are STIs!
• Serotypes A, B and C cause a serious eye
  infection that begins with conjunctivitis and may
  progress (particularly with repeated infection)
  to conjunctival scarring and blindness trachoma
• Serotypes D to K cause a less severe form of
  conjunctivitis that does not usually result in
  trachoma

Do you remember what a serotype is? see PIP!
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Trachoma

• Not an STI
• very common in tropical countries and when
  sufferers dont get treated for the initial
  infection
• transmitted via handsetc. and via flies

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C. trachomatis STIs

• The more common type of infection associated with
  serotypes D to K is sexually transmitted
• NGU (non-gonococcal urethritis) in males (also
  called NSU non-specific urethritis)
• urethritis, cervicitis, salpingitis in females
• can lead to PID (pelvic inflammatory disease) and
  resulting infertility due to scarring of
  Fallopian tubes
• also increased risk of ectopic pregnancy

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Treatment

• Azithromycin (clamelle) is usually first choice
  single dose is enough
• Alternatively can use doxycycline (adults) or
  erythromycin (babies) - Treat for extended
  periods (1-3 weeks due to prolonged replication
  cycle)

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Lymphogranuloma venereum

• C. trachomatis serotypes L1, L2 and L3 only cause
  LGV (lymhogranuloma venereum)
• begins with a genital ulcer, infection spreads to
  inguinal lymph nodes which enlarge and break
  down, discharging pus
• if untreated, can lead to enlargement
  granulomatous hypertrophy of glands

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LGV

• Was rare in developed nations before 2003
• 386 cases in UK in 2007 (500 increase in 10
  years)
• Most often seen in men who are HIV positive

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Diagnosis of Chlamydia infection

• You have two options 1) Use highly trained
  professionals or 2) Have a go yourself at home
• Which do you think is the most sensible?

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Home Chlamydia testing

• While better than no testing at all there are
  concerns that some will not follow the procedure
  correctly these tests need to be idiot proof!
• Based upon an immunochromatography test on urine
  positive colour change
• Such methods are not very sensitive so some
  positives will be missed!

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Laboratory diagnosis

• Sample type may be a swab from the affected area
  (e.g. urethra) or urine is acceptable for some
  tests
• Traditional laboratory methods include tissue
  culture assay, ELISA and immunofluorescence
• These are now being replaced by molecular assays

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Tissue culture

• Tissue Culture in cycloheximide-treated McCoy
  cells detection of inclusion bodies by iodine
  staining or IF
• Cumbersome method

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Other traditional tests

• Direct immunofluorscence using a labelled
  monoclonal antibody specific to the major outer
  membrane protein (MOMP)
• ELISA tests to detect Chlamydia antigen e.g.
  IDEIA are useful and can be automated
• However, molecular tests are rapid, specific and
  sensitive

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Molecular methods

• A number of molecular methods based on
  amplification of Chlamydia nucleic acids have
  been introduced.
• These include assays based on PCR, NASBA, TMA,
  Strand displacement amplification, LCR etc
• Most common method in UK is BD ProbeTec SDA assay
  
• www.chlamydiae.com/diagnostics_index.asp

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Syphilis

• Treponema pallidum

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Clinical and epidemiological aspects

• We have seen an 870 increase in cases of
  syphilis since 1996
• In 2007, 2680 cases were reported to HPA
• Age groups Males 25-44 years, Females 20-24
• There are hotspots of cases in the UK e.g.
  London, Manchester
• Most often seen in males, especially men who have
  sex with men (MSM)

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Numbers of diagnoses of syphilis (primary,
secondary and early latent) by sex, GUM clinics,
England and Wales, and Scotland 1931 - 2006
Equivalent Scottish data are not available
prior to 1945. N. Ireland data from 1931 to 2000
are incomplete and have been excluded.Routine
GUM clinic returns
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Number of diagnoses of infectious syphilis
(primary and secondary) by sex and male sexual
orientation, GUM clinics, United Kingdom 1997
2006
Routine GUM clinic returns
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Rates of diagnoses of infectious syphilis
(primary secondary) by sex and age group, GUM
clinics, United Kingdom 1997 - 2006
Males
Females
Routine GUM clinic returns
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Stages of disease

• There are four main stages of disease
  progressively more destructive.
• Treatment can prevent development of the next
  stage -
• 1. Primary
• 2. Secondary
• 3. Latent
• 4. Tertiary

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Clinical aspects

• Caused by the spirochaete bacterium, Treponema
  pallidum ssp pallidum
• Highly infectious
• Starts with the development of one or more ulcers
  at the point of entry of the organism CHANCRE
• A chancre is the lesion of primary syphilis
• Typically painless and will disappear even
  without treatment

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Primary syphilis

• 30 who come into contact with syphilis during
  sex will be infected
• Only 40 show symptoms of classical appearance
• 90 day incubation period
• Lesion will disappear within three weeks even
  without treatment.
• Can be missed/dismissed by patient

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Secondary syphilis

• Usually appears around 6 weeks after chancre
  disappears.
• Can be up to 2 years before signs show
• Multiple system involvement
• Mucosal and skin involvement most common
• Symptoms will resolve in most cases.
• The most infectious stage of syphilis

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Syphilis the great mimic

• The symptoms seen in patients with syphilis are
  highly variable and often similar to those seen
  in other diseases the great mimic
• Makes diagnosis without laboratory testing very
  difficult
• Sir William Osler the physician who knows
  syphilis knows medicine"

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Latent syphilis

• Two stages
• Early latent- up to 2 years
• Patient still infectious
• Late latent- after 2 years
• Patient no longer sexually infectious although
  can still pass infection vertically

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Tertiary syphilis

• 3 - 20 years after primary infection
• Benign gummatous phase - Characterized by slow
  growing granulomatous lesions
• Infiltrative or destructive
• Can affect any organ

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Tertiary syphilis

• May also see cardiovascular complications e.g.
  aortic aneurysm
• Tertiary syphilis is often associated with
  dementia CNS involvement may also present as
  general paralysis of the insane, demyelination of
  the spinal cord resulting in pains, loss of
  feeling and difficulty walking. Changes in the
  joint - so-called Charcot's joints may develop
  owing to loss of nerve supply

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Congenital syphilis

• If infection is acquired in pregnancy, usually
  miscarriage or still-birth ensues. However, if
  the foetus survives, it may show signs of
  congenital syphilis the Hutchinsons Triad
  Hutchinsons teeth (pointed), deafness
  keratitis
• There is a statutory requirement to screen all
  pregnant women for evidence of syphilis
  antibody test (see later)

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Treatment

• Syphilis is a potentially devastating disease
  that is easy to treat, but it is essential that
  it is caught in the early stages.
• Benzathine penicillin is usually used. A single
  dose is sufficient to cure primary syphilis,
  although longer treatments are required for later
  stages, including the treatment of late latent
  syphilis. No penicillin resistance has been
  observed

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Treponema pallidum ssp pallidum

• Treponema pallidum ssp pallidum is a very long,
  slender bacterium, which is about 0.1µm in
  diameter and 22µm in length
• Since the maximum resolution of a bright-field
  microscope is 0.2µm, the organism cannot be seen
  by conventional microscopy
• Cannot Gram stain this organism

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T. pallidum as seen by EM
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Treponema pallidum ssp pallidum

• Can we culture this organism using artificial
  media?
• NO!
• The organism has undergone reductive evolution so
  it has lost many of the metabolic processes
  required for independent growth
• This rules out using culture and identification
  as a diagnostic tool

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Other subspecies

• There are three other subspecies of T. pallidum
  these cause the non-venereal infections yaws,
  pinta and bejel
• These are found in the Caribbean and W. Africa
  they are now very rare
• However, we need to bear these in mind as the
  antibody response to syphilis is identical to
  these 3 infections
• Potential for misdiagnosis when interpreting
  serology results!

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Laboratory diagnosis

• Diagnosis is usually confirmed using both
  clinical evidence and laboratory test results
• Can we see syphilis down the microscope?
• YES using dark ground microscopy
• What are the disadvantages of this test?

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T. pallidum by dark ground microscopy
66
Dark ground microscopy

• Usually done in Genitourinary medicine clinics
• Take fluid from an abraded ulcer view sample
  against a dark background
• Treponema is apparent by virtue of refractivity
• Also often see characteristic corkscrew motility

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Serology

• Can detect an antibody response to infection
  using serology
• A major disadvantage of serology is the immune
  system takes a while to produce antibodies so
  early infection will be missed
• There are a number of serological tests for
  syphilis BUT no one method is 100 reliable
• This makes the interpretation of serological
  tests a bit tricky (but I will explain)

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Serological tests

• Serological tests for syphilis can be divided
  into two general types
• Non-specific tests these rely on the fact that
  syphilis antibodies also bind (cross-react) to
  cardiolipin (found in ox heart) e.g. VDRL and RPR
  tests
• Specific tests e.g. TPPA (TPHA), ELISA and FTA
  (abs)
• If positive with one method must confirm with a
  second method

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Venereal Disease Reference Laboratory test

• Mix patient sera with antigen (cardiolipin) on a
  slide for 8 mins
• Examine for agglutination (positive test)
• Quantitative test if positive test a dilution
  series of sera to obtain the highest dilution
  which is positive antibody titre

Positive
Negative
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VDRL

• VDRL becomes positive 1-2 weeks after chancre
  appearance (73) and reaches high titres in
  secondary syphilis (100)
• BUT becomes negative in latent syphilis and also
  following treatment
• Therefore this test is very important in
  monitoring the effect of treatment and
  stage/activity of disease
• False positives are a problem (e.g. recent
  vaccination, connective tissue disease)

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Treponema pallidum Particle Agglutination test
(TPPA)

• Specific test for syphilis antibodies
• Patient sera diluted in a microtitre plate
• Gelatin particles control particles
• Gelatin particles coated with Treponema antigen
  test particles
• Added to different wells
• Incubate
• Observe for agglutination indicates serum
  antibodies reacting with antigen on particles
• If negative the particles will sink to the bottom
  of the well

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TPPA
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TPPA

• Becomes positive in primary syphilis (71) and
  100 positive in secondary
• Remains positive for life even if treated
• Test used to be performed using sheep
  erythrocytes not gelatin particles TPHA
• TPPA is a cumbersome test to perform so used as a
  confirmatory test
• For screening patients (e.g. in pregnancy) we use
  an ELISA test - automated

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ELISA

• Some ELISA kits detect IgG only (OK), others
  detect IgM IgG (best as helps determine stage
  of disease)
• Positive in 82 of cases of primary syphilis and
  100 of secondary
• Remains positive despite treatment (IgG)
• If positive confirm usually with TPPA test

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FTA (abs)

• Indirect immunofluorescence test
• Gold standard test positive in 86 of primary
  syphilis and 100 of secondary
• However it is a cumbersome and difficult test to
  do so it is only performed in reference
  laboratories

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Laboratory diagnosis of syphilis

• If patient presents with an ulcer perform dark
  ground microscopy if positive begin treatment
  and monitor by serology
• If no ulcer or microscopy is negative we must
  rely on serology
• ELISA is used as a screening test as it is cheap,
  automated and rapid
• If positive perform a TPPA to confirm a true
  positive.

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Laboratory diagnosis of syphilis

• If positive by ELISA and TPPA begin treatment and
  perform VDRL test to ensure patient is clear from
  infection
• What laboratory results would you see in a case
  of secondary syphilis?
• Or latent syphilis?