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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GonorrhoeaNeisseria gonorrhoeae
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Clinical and epidemiological aspects

• 2nd commonest bacterial STI
• 2004 22,335 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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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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• Both sexes disseminated infection on rare
  occasions septic arthritis, pustular rash, even
  infective endocarditis
• Infection during pregnancy ophthalmia
  neonatorum of baby (conjunctivitis)
• Dual genital infection with Chlamydia trachomatis
  usual to treat for both at time of gonorrhoea
  diagnosis

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Microbiological aspects

• 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 commensal species of
  Neisseria, often found in the pharynx

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• N. gonorrhoeae is phagocytosed by
  polymorphonuclear neutrophils (pus cells) but
  resists intracellular destruction, remaining
  intact within the pus cell.
• Readily cultivable, although it is sensitive to
  desiccation and requires aerobic incubation with
  5 to 10 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 and showing colonial
  variation. 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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Diagnostic Tests for Gonorrhoea

• For urethral, cervical and rectal infections,
  microscopy (Gram strain) is a very useful
  investigation
• Gram-negative diplococci, with a coffee bean
  shape, are looked for within poymorphonuclear
  neutrophils (PMN)
• For pharyngeal infections, microscopy is of no
  value because of the presence of commensal
  Gram-negative diplococci in the throat

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Intracellular, Gram-negative diplococci - N.
gonorrhoeae
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• Culture is mandatory - for identification and
  antibiotic sensitivity tests
• Selective medium containing antibiotics and
  growth supplements (look this up)
• e.g. Thayer Martin or New York City
• PCR tests have been developed for the detection
  of N. gonorrhoeae infection and a single swab may
  be used in a double test to detect N. gonorrhoeae
  and Chlamydia trachomatis.

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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 monoclonal antibody
• API NH utilizes carbohydrates plus enzymes
  activity, similar to API 20E

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Treatment

• The recommended treatment of gonorrhoea is
  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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ChlamydiaChlamydia trachomatis
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ChlamydiaClinical and epidemiological aspects

• The most common bacterial sexually transmitted
  infection, with 104,155 cases reported to the
  Health Protection Agency in 2004
• The causative organism is Chlamydia trachomatis
• The number of cases has risen steadily since the
  mid 1990s

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• The infection has a longer incubation period than
  gonorrhoea, of 1 to 3 weeks
• Asymptomatic infection is common in both sexes
  at least 50 in males and 70 in females
• The highest rates of infection occur in young
  people under the age of 24

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Signs and symptoms

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

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• 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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• 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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• Two other species of Chlamydia are known to be
  pathogenic for humans
• C. pneumoniae is a cause of pneumonia and one
  of the agents of atypical pneumonia
• C. psittaci is a respiratory pathogen in
  psittacine birds, such as parrots. The infection
  (a zoonosis) is transmissible to man, and may
  cause a severe pneumonia

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Life cycle 1

• obligate intracellular bacterial pathogen
• all chlamydiae undergo a similar life cycle
  within the cell they are infecting
• Elementary 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 2

• Attachment
• 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 3

• Reticulate Body Formation
• 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 4

• Staining of the Inclusion Body with iodine
  todemonstrateinfection of cellcultures

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Life cycle 5

• 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

• Many different serotypes and these can be grouped
  according to the type of disease that they cause
• 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

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Trachoma

• 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
  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 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

• Serotypes L1, L2 and L3 cause LGV (lymhogranuloma
  venereum)
• begins with a genital ulcer,next inguinal lymph
  nodesenlarge and break down,discharging pus
• if untreated, can lead toenlargement
  granulomatoushypertrophy of glands

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Diagnosis of Chlamydial Infection

• Because of the absolute requirement for a host
  cell Chlamydia cannot be grown on agar based
  culture media other methods used
• Direct immunofluorescence using monoclonal
  antibodies
• Tissue Culture in cycloheximide-treated McCoy
  cells detection of inclusion bodies by iodine
  staining or IF
• ELISA for chlamydial antigen detection
• Nucleic acid amplification tests (NAAT)
• polymerase chain reaction (PCR)
• ligase chain reaction (LCR)
• strand displacement amplification (SDA)
• transcription mediated amplification (TMA)
• For descriptions, please see
• www.chlamydiae.com/diagnostics_index.asp

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• Syphilis
• Treponema pallidum

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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

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Primary syphilis
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• Appears up to 3 weeks post exposure
• Heals in 2 to 6 weeks
• If untreated, 40 of patients go on to develop
  secondary syphilis
• 2º syphilis develops 2-6 weeks after appearance
  of a chancre
• Rash including palms/soles, lymphadenopathy,
  flu-like symptoms
• If still untreated, 2º syphilis may be followed
  by tertiary syphilis

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Secondary syphilis
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• Tertiary syphilis develops 4 or more years after
  untreated primary syphilis
• Tertiary syphilis (late syphilis) may affect many
  parts of the body
• Characterized by slow growing granulomatous
  lesions which affect the nervous system, leading
  to general paralysis of the insane and
  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.
  Dementia may occur. Very rarely changes in the
  aorta may result in an aneurysm.

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Tertiary syphilis
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• The other 60 of untreated cases will develop
  latent syphilis
• Latent syphilis may reactivate at any time to
  give rise to symptoms of later stages of 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

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• There is a statutory requirement to test all
  pregnant women for anti-treponemal antibodies, so
  as to reduce the likelihood of congenital
  syphilis.
• Syphilis is a potentially devastating disease
  that is easy to treat, but it is essential that
  it is caught in the early stages. Treatment of
  later stages of the disease will halt its
  progress but not reverse any damage that has
  already occurred.

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• There are other treponemal diseases, known as
  non-venereal treponematoses, which have to be
  born in mind when interpreting diagnostic tests
  for antibody. These are yaws, bejel and pinta.
  They are not endemic in the UK, but yaws is a
  common infection of childhood in parts of Africa
  and the West Indies.

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Treatment of syphilis

• Benzathine penicillin, given by intramuscular
  injection, is the first line treatment for
  syphilis. A single dose is sufficient to cure
  primary syphilis, although longer treatments are
  required for later stages, including the
  treatment of late latent syphilis.
• Alternative drugs for penicillin-hypersensitive
  patients are ceftriaxone (injection) or
  doxycycline (oral).

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Epidemiology of syphilis

• In 2004 there were 2,254 cases of syphilis
  diagnosed in sexual health clinics in the UK
• 870 increase since 1996!
• Most common in males aged 20-24 years, females
  aged 25-34 years
• Syphilis was most prevalent during the late
  1940s, but a resurgence occurred around the
  beginning of this century

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Microbiological Aspects

• 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
  in a Gram-stained slide
• Cannot be grown in artificial culture

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Treponema pallidum ssp pallidum(electron
micrograph)

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• The non-venereal treponematosis yaws is caused
  by T. pallidum ssp pertenue, bejel by T. pallidum
  ssp endenicum (also termed endemic syphilis) and
  pinta by T. carateum

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Diagnosis of syphilis

• Since it cannot be grown in vitro, microscopy or
  serology are used
• What are the disadvantages of these?
• Dark ground microscopy of fluid taken from an
  abraded ulcer treponemes apparent by virtue of
  refractility
• The organisms show cork-screw motility
• Clinical information is v important

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Treponema pallidum ssp pallidum by dark ground
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Serological Tests

• Disadvantages
• an antibody response does not appear until about
  one week after the appearance of a chancre
• No one test is 100 reliable for 1 syphilis
• there is no way of distinguishing serologically
  between syphilis and the non-venereal
  treponematoses

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• The tests may be grouped into 2 types specific
  tests and non-specific tests, but both types have
  enormous value in diagnosis all positive tests
  require confirmation using another method
• Non-specific tests
• Rapid plasma reagin (RPR) test
• Venereal Diseases Research Laboratory (VDRL)
  carbon test
• Employ cardiolipin as antigen
• Alcoholic extract of ox heart

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• Serum is mixed with antigen on a card
• Rotation for 8 minutes
• Examine for agglutination
• Double dilutions of positive sera to obtain a
  titre
• Monitors treatment and activity of disease
• Becomes positive 1-2 weeks post chancre
  appearance, high titre in 2º syphilis
• Becomes negative after treatment
• Biological false positives rheumatoid disease,
  viral pneumonitis, post vaccination, pregnancy

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Treponemal (specific) tests

• Treponema pallidum Particle Agglutination (TPPA)
• Enzyme-Linked Immunoassay (ELISA)
• Fluorescent Treponemal Antibody (Absorbed)
  (FTA(Abs))

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TPPA

• Serum diluted in microtitre plate
• Gelatin particles control particles
• Gelatin particles coated with treponemal antigen
  test particles
• Added to different wells
• Incubate
• Observe for agglutination

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• TPPA is positive in 60 of cases of primary
  syphilis, 100 of cases of secondary syphilis
• Remains positive for many years despite treatment
• Very specific for treponemal diseases
• False positives glandular fever, leprosy,
  Systemic lupus erythematosus

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ELISA

• If an ELISA is used which detects both IgM and
  IgG antibody classes, this is an excellent screen
  test (can be automated) and will be positive at
  all stages of disease
• Becomes positive 1 week after chancre develops
• Remains positive despite treatment - IgG
• Requires confirmatory test (e.g. TPPA)

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FTA (Abs)

• Indirect immunofluorescence test
• Spot slide coated with treponemes
• Serum absorbed (to remove non-specific treponemal
  antibody)
• Serum added to spot, incubated, washed, dried
• Anti-human globulin plus fluorescein added
  binds to antibody coating treponemes
• Incubate, wash, dry slide, observe by UV
  microscopy for fluorescence

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• Seen as the gold standard test but used only in
  specialist laboratories
• Becomes positive 1 week after development of a
  chancre, remains positive for years but becomes
  weaker in time
• Strongest reaction when RPR is positive, i.e.
  when disease is active
• False positives can occur in herpetic infection
  and in rheumatoid disease

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Example 1

• VDRL negative
• TPHA negative
• ELISA negative
• Either negative or very early disease
• Repeat if at risk

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Example 2

• Positive VDRL
• Negative TPHA
• Negative ELISA IgG
• FALSE POSITIVE

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Example 3

• VDRL positive
• TPHA positive
• ELISA positive - IgM
• Recent/Active syphilis infection

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Example 4

• ELISA positive- IgG
• TPHA positive
• VDRL negative
• Treponemal infection at some time-either treated
  or untreated latent/tertiary