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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
Rates of diagnosis of uncomplicated gonorrhoea by sex and age group, GUM clinics, United Kingdom: 1997 - 2006 Routine GUM clinic returns Males Females
Number of diagnoses of uncomplicated gonorrhoea by sex and male sexual orientation, GUM clinics, United Kingdom: 1997 - 2006 Routine GUM clinic returns
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
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
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
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.
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
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
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
Chlamydia Chlamydia trachomatis
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
Rates of diagnoses of uncomplicated genital chlamydial infection by sex and country, GUM clinics, United Kingdom: 1997 - 2006 Routine GUM clinic returns Males Females
Rates of diagnoses of uncomplicated genital chlamydial infection by sex and age group, GUM clinics, United Kingdom: 1997 - 2006 Routine GUM clinic returns Males Females
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
Government screening for Chlamydia in under 25’s announced in 2003
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?
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
Symptoms (when present)
unusual vaginal discharge
pain on urination
lower abdominal pain
burning and itching in genital area
pain on urination
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 Reiter’s Syndrome (arthritis)
In females, the consequences of untreated infection are pelvic inflammatory disease (PID) in 10 to 40% of cases
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
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?
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 0.3 μ m in diameter. EBs are fairly inert and can survive outside the cell
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
the EB develops into a Reticulate Body (RB) which is larger (0.5 to 1.0 μ m) 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”
Staining of the Inclusion Body with iodine to demonstrate infection of cell cultures
EB Formation and Release
after 18 to 24 hours, the RB reorganises into many EBs which are released on cell rupture (24 to 48 hours after infection)
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!
Not an STI
very common in tropical countries and when sufferers don’t get treated for the initial infection
transmitted via hands etc. and via flies
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
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)
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
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
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?
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!
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
Tissue Culture in cycloheximide-treated McCoy cells – detection of inclusion bodies by iodine staining or IF
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…
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
Syphilis Treponema pallidum
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)
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
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
Rates of diagnoses of infectious syphilis (primary & secondary) by sex and age group, GUM clinics, United Kingdom: 1997 - 2006 Routine GUM clinic returns Males Females
Stages of disease
There are four main stages of disease – progressively more destructive.
Treatment can prevent development of the next stage -
Caused by the spirochaete bacterium, Treponema pallidum ssp pallidum
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
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
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
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…"
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
3 - 20 years after primary infection
Benign gummatous phase - Characterized by slow growing granulomatous lesions
Infiltrative or destructive
Can affect any organ
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
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 Hutchinson’s Triad – Hutchinson’s teeth (pointed), deafness & keratitis
There is a statutory requirement to screen all pregnant women for evidence of syphilis – antibody test (see later)
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
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
Treponema pallidum ssp pallidum
Can we culture this organism using artificial media?
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
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!
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?
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
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…)
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
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
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)
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
Observe for agglutination – indicates serum antibodies reacting with antigen on particles
If negative the particles will sink to the bottom of the well
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
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
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
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.
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?