Download HSV-2

Why STI Diagnosis is
Important
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•
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STIs rapidly spread throughout communities.
STIs can be associated with acute illness.
STIs can be associated with chronic illness.
STIs can be associated with remote illness.
Ulcerative STI associated transmission of
other illnesses, especially HIV.
GENITAL ULCER DISEASE (GUD)
I. GENERAL CONSIDERATIONS
Genital ulcer disease is one of the major STD
syndromes.
SETTING UP RAPID, SIMPLE, EASY TO
INTERPRET TESTS IS THE CHALLENGE IN
GUD DIAGNOSIS
DIFFERENTIAL DIAGNOSIS (I)
A. STD-related etiologies and organisms:
1. Genital herpes: Herpes Simplex Virus Type 1 and Type 2
2. Primary syphilis: Treponema pallidum
3. Chancroid: Haemophilus ducreyi
4. Lymphogranuloma venereum (LGV): Chlamydia
trachomatis
5. Granuloma inguinale (Donovanosis): Calymmatobacterium
granulomatis
DIFFERENTIAL DIAGNOSIS (II)
B. Non STD-related etiologies:
1. Non-STD infectious causes of GUD: scabies, common
skin infections (e.g. Staph).
2. Non-infectious causes of GUD: aphthous ulcers,
Behcet’s syndrome, fixed drug eruption, Reiter’s syndrome,
trauma/abrasions.
C. No etiology
No etiology is found in 20% to 50% of GUD cases, most
likely related to the sensitivity of the laboratory tests
(affected by self-medication, duration of lesion, technology
of the test).
EPIDEMIOLOGY (1)
1. It is important to know the epidemiological
risk factors of disease, including
demographic and behavioral characteristics of
the patient,
and travel abroad or in regions with high rates
of syphilis or chancroid.
EPIDEMIOLOGY (2)
2. Globally, the most frequent cause of STDrelated GUD is genital herpes, followed by
syphilis, then chancroid.
Lymphogranuloma venereum (LGV) is rare in
the U.S. and Europe, and granuloma inguinale
(GI, or donovonosis) is almost never
encountered in the U.S. and Europe
3. In developing countries, leading causes
of GUD are infections with H. ducreyi,
followed by infections with T. pallidum and
HSV infections.
4. More than one disease is sometimes
present in a patient with genital ulcers.
EPIDEMIOLOGY (3)
5. Multiple studies have demonstrated that the
presence of GUD increases the risk of HIV
infectiousness and susceptibility, resulting in an
estimated 2-5-fold increase in HIV transmission
rate with GUD.
Significant associations with chancroid and syphilis were
reported, but the main association was with HSV-2
Active coinfection with HIV-1 and HSV-2 seems to
accelerate the progression to AIDS.
Also, the transmission of HIV-1 may be facilitated
because an increased HIV-1 viral load in blood, plasma,
and semen was found in the presence of genital ulcers
due to HSV-2
BIBLIOGRAFY
Dickerson MC, Johnston J, Delea TE, White A, Andrews E.
The causal role for genital ulcer disease as a risk factor for
transmission of human immunodeficiency virus. An application of the
Bradford Hill criteria. Sex Transm Dis. 1996 Sep-Oct;23(5):42940.
Moodley P, Sturm PD, Vanmali T, Wilkinson D, Connolly C,
Sturm AW. Association between HIV-1 infection, the etiology of
genital ulcer disease, and response to syndromic management. Sex
Transm Dis. 2003 Mar;30(3):241-5.
J McClelland RS, Lavreys L, Katingima C, Overbaugh J, Chohan
V, Mandaliya K, Ndinya-Achola J, Baeten JM. Contribution of
HIV-1 infection to acquisition of sexually transmitted disease: a 10year prospective study. Infect Dis. 2005 Feb 1;191(3):333-8.
DIAGNOSTIC APPROACH (1)
A. Patient history:
1. Lesion history: prodrome, initial presentation (especially presence
of vesicles), duration of lesion, pain, other systemic symptoms, use of
systemic or topical remedies, any history of similar symptoms in the
past or partners with similar symptoms.
2. Medical history: HIV status, skin conditions, drug allergies,
medications.
3. Sexual history: gender of partners, number of partners (new,
anonymous, serodiscordant), venue for meeting partners, commercial
sex exposure, partners with symptoms or signs, partners with known
HSV or recent syphilis diagnosis.
4. Travel history
DIAGNOSTIC APPROACH (2)
B. Physical exam:
1. Lesion: examine for appearance, distribution,
number, size, induration and tenderness.
2. Genital exam: examine genital and perianal area
for other lesions.
3. Lymph node(s): note number and location of
enlarged nodes, size,tenderness.
4. General exam: thorough examination of oral cavity
and skin of torso, palms and soles, and neurologic
exam, including cranial nerves.
LESIONS
Genital ulcers may present themselves in various forms
The classic lesion of primary syphilis is a single, painless,
indurated ulcer with a clean base and is caused by active
infection with T. pallidum
The herpetic lesion is characterized by multiple painful
lesions which may be recurrent
There is, however, significant variability in morphologic
presentation, making the clinical interpretation unreliable
when used without confirmatory laboratory tests
DIAGNOSTIC APPROACH (3)
C. Laboratory testing:
1. General approach. After a sexual history and physical
exam of the patient presenting with GUD, the provider
needs to consider the complete differential diagnosis and
conduct laboratory testing based on clinic testing capability
and probability of disease.
a) ALL patients with GUD should receive a non-treponemal
syphilis serology test (RPR or VDRL).
b) If HSV is suspected, a culture or antigen test for HSV should
be performed. Consider HSV type-specific serology if
history is suggestive of a recurrent lesion or no lesion
amenable to culture.
..laboratory testing
c) All patients with sexual risk factors presenting with
GUD should be offered counseling and testing for HIV, and
screened for other STDs (e.g. chlamydia and gonorrhea).
d) Routine testing of all patients with GUD for chancroid is
not indicated. Consider if the patient gives a history of
travel to an area where chancroid is prevalent, or if the
lesion does not respond to treatment, in a patient with
negative syphilis serologies.
Different laboratory tests can be used to
discriminate between the causative agents of GUD,
and each test differs with respect to sensitivity,
specificity, and turnaround time
Diagnosi di laboratorio
-Diagnosi diretta
-Diagnosi indiretta
-importante per HSV
-fondamentale per T.pallidum
DIAGNOSI DIRETTA
INFORMAZIONI
GENERALI
HSV
Culture provides direct evidence for infection and is
the "gold standard" for HSV detection, but it may
take up to 1 week to get a definitive negative result.
Positive results can be obtained within 2 days, and the
differentiation into HSV-1 or HSV-2 is possible by
using monoclonal antibodies.
Direct detection of HSV antigen by immunoassay also
enables a fast diagnosis.
T. PALLIDUM
In vitro culturing of T. pallidum is not possible
at all.
T. pallidum can be detected by dark-field
microscopy examination, but this is a
specialized test that is not routinely performed.
H. DUCREY
H. ducreyi is a fastidious microorganism that
is detected by a rather problematic culture
technique. Nevertheless, culture is the gold
standard for H. ducreyi detection.
Polymerase chain reaction
In the past decade, amplification techniques such as
PCR have been developed to detect many different
infectious agents, including HSV-1, HSV-2, T. pallidum,
and H. ducreyi.
PCR can be performed for each agent separately or,
more efficiently, by a multiplex assay.
The advantages of PCR are the direct detection of the
pathogen itself, the high sensitivity, and the
potentially short turnaround time.
A disadvantage is that it should be performed with
great care to prevent carryover contamination.
Advantages of PCR in GUD
diagnosis
PCR represents a great improvement for laboratory diagnosis in the
sense that there are fewer patients with genital ulcers for whom no
definite cause is found.
The turnaround time is shortened from up to 1 week for herpes virus
culture to 1 to 3 days for PCR.
The PCR is significantly more sensitive than culture of HSV
T. pallidum PCR performed early in infection is more informative
than usually used serological test (see later).
A positive PCR result is indicative of an active H. ducreyi infection.
So, when the costs of diagnosis are comparable and the PCR can be
performed on a regular basis (i.e., three times per week), PCR
detection will become the gold standard for GUD diagnosis.
Herpes genitale
Pathogenesis of genital
HSV infection
Primary infection
with HSV
(Sexually transmitted)
Viral shedding
HSV replication in
genital mucosa or skin
HSV replication in
genital mucosa
Uptake of HSV by
sensory nerves with
retrograde transport
to sensory ganglia
Host Immune Response:
Circulating and Mucosal
Antibody production
Cytokine production
Cell-mediated immunity
Anterograde transport of virus to
mucosal and cutaneous genital
tissues
Productive viral replication in
sensory neurons
Dorsal
root
ganglia
Limits viral replication at
all sites
Establishment of nonreplicating, latent infection in
sensory neurons
Dimensioni del problema
Ogni anno circa 200.000-500.000 americani, in
maggioranza adolescenti e giovani, sviluppano l'infezione
primaria da virus Herpes simplex genitale (HSV)
Un numero di individui compreso tra 25 e 31 milioni è
inoltre cronicamente infetto
Entrambi i ceppi di HSV, il tipo 1, HSV-1 e il tipo 2, HSV-2,
possono causare malattia a livello genitale, ma è l'HSV-2 che
provoca la maggioranza delle infezioni genitali recidivanti.
A woman had a normal course of pregnancy. In particular, there were no
clinical and laboratory hints to rubella, varicella, syphilis and the acquired
immunodeficiency syndrome. The women did not give a history of genital
lesions, perineal changes and dysuria during or prior to pregnancy.
She was admitted to the hospital since she developed fever, rupture of fetal
membranes and premature labor at the 28th week of gestation.
At the time of delivery, there were no active herpes lesions.
The maternal group B streptococcus (GBS) status was negative. An antibiotic
therapy and treatment for fetal lung maturation using betamethasone were
started. Because of increasing infection parameters and therapy-resistant labor,
the baby was born after 30 + 2 weeks of gestation by Caesarian section.
Since the boy developed progressive respiratory insufficiency and
had a high oxygen demand of up to 100%, he had to be intubated and
supplied with oxygen at the age of 5 h.
The laboratory parameters did not indicate infection.
However, the placental data revealed histopathological evidence of
acute Chorioamnionitis.
The chest X-ray on the first day of life revealed signs of respiratory
distress syndrome degree III. Because of the negative maternal GBS
status, a neonatal GBS pneumonia could be excluded. …………
continuous positive airway pres-sure …………..oxygen for several
months …..radiograph of chest indicated broncho-pulmonary
dysplasia.
At the age of 6 weeks, the ophthalmologic examination, showed a
chorioretinal scar in the right eye. Laboratory studies to detect CMV in
urine and blood using PCR were negative as were Toxoplasma gondiispecific Ig G and IgM antibodies.
From the age of 4 months, the infant developed vesicles on the nose
suggestive of recurrent herpes simplex infection.
Using PCR, HSV-2 DNA was detected in the secretion of vesicles, but
the HSV-2 strain was not isolated in cell culture.
The HSV type-specific serostatus using HerpeSelect® 1 and 2 ELISA
IgG and recomLine HSV-1 & HSV-2 IgG was determined in the mother
and the infant. HSV-2 IgG was positive and HSV-1 IgG was negative
in both suggesting previous HSV-2 infection.
This means that in the immunoblot both the HSV lysate band and the
gG-2 band reacted with positive whereas the gG-1 band was
assigned as negative.
As the present case demonstrates, prenatal HSV infections may not to
be associated with severe congenital abnormalities and can be
overlooked easily.
They cannot be diagnosed if suitable methods are not used or are not
available for routine diagnostics.
In the case presented, the chorioretinal scar detected in the 6-week-old
infant was taken as an opportunity to exclude congenital T. gondii and
CMV infections in the neonate. Although the results were negative,
the neonatologists omitted further microbiological diagnostics.
The retrospective type-specific serologic diagnosis revealed a previous
HSV-2 infection of the mother resulting most likely in a prenatal HSV-2
infection of the infant.
Using these methods, the mother was identified to be at risk of
infecting her fetus.
Most likely, the mother had a primary HSV-2 infection shortly
before the delivery and infected her fetus.
The histopathologi-cal evidence of chorioamnionitis indicated
ascending infection.
Even though the woman could not remember symptomatic genital
herpes during pregnancy, she was admitted into the hospital
because of a febrile illness accompanied by a premature labor.
Case reports of intrauterine HSV infection suggest an active
maternal HSV lesion usually referred to as primary lesion or a
significant maternal febrile illness in pregnancy as the cause
of intrauterine infection.
Additionally, primary infection goes unnoticed in more than half of
the patients
In the present case, it can be speculated whether the
premature birth and the respiratory distress syndrome were
also caused by HSV-2.
A recent seroprevalence study of HSV-2 in Germany showed that
between 2.7% and 4.7% of all children aged up to 15 years
possess HSV-2-specific antibodies probably acquired by an
intrauterine or neonatal infection which may be clinically
unapparent or not recognized clinically.
E IN ITALIA?
- Suligoi et al. Sex. Trasm. Inf., 2000
-Cusini M et al. Sex. Trasm. Dis., 2000
Sieroprevalenza per HSV-1 e HSV-2 in tre
città italiane
Studio di sieroprevalenza HSV, 2000-01
2.014 soggetti della popolazione generali di Genova, Roma
e Lecce
HSV-1
positivi
HSV-2
71%
positivi
71%
9%
9%
negativi
negativi
Sieroprevalenza
dell’HSV-2 in
adulti: Italia
(987 soggetti di età> 20
anni della popolazione
generale)
15,0%
5,5%
Studio IHF
Sieroprevalenza HSV-1 e
HSV-2, 2000-01
9,9%
HSV-2 seroprevalence by age
and gender in a MST clinic, Milan
Study on 919 patients, 661 males and 258 females
% HSV-2 +
50
45
40
35
30
25
20
15
10
5
0
men
women
total
Age in years
<=25
26-35
>35
Diagnosing Genital Herpes How Do You Do It?
Steps to a Diagnosis
There are two general approaches to diagnosing
genital herpes:
•Physical exam and history
•Laboratory tests
WHAT LABORATORY TESTS?
A visual examination is only one part of a complete
diagnostic work-up.
Making a diagnosis of genital herpes by visual inspection and
by asking questions is not easy and even experts can be
wrong.
For one thing, genital herpes does not look the same in
every patient; it can "mimic" the appearance of other sexually
transmitted diseases (STDs) and other STD's can look like
herpes.
A mild case of herpes can easily be mistaken for something
that has nothing to do with STDs such as a simple rash.
Thus confirming a diagnosis with a laboratory test is
critical.
Manifestations of disease among
individuals infected with HSV-2
20%
Asymptomatic
20%
Recognized
symptomatic
60%
Unrecognized
symptomatic
LA DIAGNOSI DI INFEZIONE GENITALE DA HSV
SI PUO’ OTTENERE SOLO QUANDO LA
VALUTAZIONE CLINICA SIA ACCOMPAGNATA
DALLA COLTURA VIRALE E DA TEST DI
SIEROLOGIA TIPO-SPECIFICA

EPISODIO PRIMARIO O RICORRENZA?
TIPO DI VIRUS?

CORRETTO MANAGEMENT
DIAGNOSI DIRETTA:
Messa in evidenza di HSV -1
e -2 o di parti di questo
(antigeni o acidi nucleici)
Test di Laboratorio per la
Diagnosi Diretta
TEST
Isolamento virale
SENSIBILITA’/SPECIFICITA’
Alta (>90%)
Alta
(esame colturale)
Rilevazione di Antigeni
(immunofluorescenza su
Bassa
Alta
vetrino, EIA)
Rilevazione di Acidi
Nucleici
Molto elev
Molto elev
Laboratory Tests
Testing for the virus directly from the skin is useful if
genital signs or symptoms are present at the time
you are examined.
When lesions or sores are present the physician or
health care provider can rub the sore with a swab and
submit the sample for detection of herpes simplex
virus.
There are four main ways laboratories detect HSV
from swabs:
1. Viral Culture
HSV-1 & HSV-2 grow in what is called tissue culture cells or
"cell culture." When changes in the cell culture are seen under a
microscope, the laboratory does further tests to show that the
changes are due to HSV and that the virus is HSV-1 or HSV-2.
This picture was taken through a microscope to show cells
that have rounded up due to HSV infection. Isolation of
HSV from a sore by growth in cell culture is definitive
proof that HSV caused the sore.
However…
Viral culture can take 1-10 days to become
positive and even a good sample taken from a
lesion with herpes may be negative
Some laboratories use a version of viral culture
called "shell vial" culture to make the test
faster (only 1-2 days) but this test is not as
sensitive as standard viral culture.
This means the shell vial test has a higher
chance than standard culture of being negative
even though herpes is present.
ELVIS-ID TEST
An interesting version of viral culture is called the ELVISTM
(enzyme-linked virus-inducible system) -Id test.
This test uses cells containing a HSV-specific gene promoter
sequence linked to the reporter gene b-galactosidase.
When HSV from a patient's swab infects the ELVIS cells, this
enzyme is switched "on" and causes the cells to turn blue.
This photo shows ELVIS "blue cells" surrounded by uninfected
clear cells.
DISADVANTAGES
ELVIS tests are usually completed in 1-2 days
BUT:
It is more expensive than a standard culture test
It may miss some cases of herpes either because there is so
much virus in the sample that the cells are destroyed
before the test can be completed or because there is very
little herpes in the sample
2. HSV Fluorescent Antibody Test
("HSV FA")
Fluorescent antibody techniques (FA) are faster than
culture and nearly as sensitive for lesions that have newly
formed.
These tests are less accurate for older, healing lesions.
Occasionally, FA can be more sensitive than culture if
the virus has not survived transport.
3. Diagnosi citologica
• Cellule ottenute da scraping delle
lesioni viene strisciato su vetrino
• Fissazione in etanolo freddo
• Colorazione secondo Papanicolaou,
Giemsa (Tzanck) o Wright
• Ricerca di cellule giganti
multinucleate con inclusioni
intranucleari: sensibilità 60-70%
4.Rilevazione di Acidi
Nucleici: PCR (I)
• Rappresenta il metodo diagnostico di scelta
nelle infezioni erpetiche del SNC
• Questo procedimento in condizioni ottimali
dimostra una sensibilità del 95-100% ed
una specificità del 97-100%
• Permette la tipizzazione virale con la
scelta di primer opportuni
• Richiede piccole quantità di materiale
clinico
• Test rapido (poche ore)
4.Rilevazione di Acidi
Nucleici: PCR (II)
SVANTAGGI:
•I controlli per le cross-contaminazioni sono
fondamentali
•Scarsa disponibilità di saggi commerciali
•Alti costi
•Richiede laboratori attrezzati
•Personale tecnico con esperienza di biologia
molecolare
Should I ask for a PCR or a culture?
At this time, PCR is less available and more
expensive than culture or FA.
But PCR is more likely to give a correct
answer.
The chance of missing herpes in a lesion by
culture or FA is much higher than with PCR.
If PCR is not affordable or available, a type
specific serology blood test is good
alternative to detect HSV infection.
The New Blood Tests ("Type-Specific
Serology")- An Overview
The most important breakthrough in herpes diagnosis in the last
few years is the development of serology tests that
accurately tell you if you are a carrier of HSV-2.
Serology tests detect "antibodies" in the blood.
Blood tests obviously do not require swabbing a lesion, so
they can be done long after symptoms have faded.
The key to accuracy for herpes blood tests is to make sure that
the test is a 'type-specific' assay that can tell the difference
between HSV-1 and HSV-2 antibodies.
Many commercially available tests currently can
not make this distinction.
Type-specific antibodies
The new blood tests are based on antibodies to two
proteins that are part of the HSV-1 and HSV-2 virus
envelope.
•One protein is called glycoprotein gG-1 and it is found
only on the outside of the HSV-1 virus or in cells infected
with HSV-1 as the virus is produced.
•The other protein is called glycoprotein gG-2 and is
found on HSV-2 virus or in cells infected with HSV-2.
These proteins have similar names but they are enough
different that antibodies that are produced to one HSV
type (say, HSV-1) can not be mistaken for the other
type (HSV-2) in the new serology tests.
We call antibodies that can react to only one, not both,
HSV types "type specific."
Isn't gG a kind of antibody?
The short name for "glycoprotein G" is a common source of
confusion. The main antibody to develop in response to
infection is called Immunoglobulin G or "IgG".
When scientists were working out the structure of herpes
virus, they named the viral glycoproteins alphabetically in
order of their discovery.
Thus, gG was the seventh glycoprotein to be discovered.
As shown in the diagram, other glycoproteins such as gB and
gD are also on the outside of the virus.
Metodi sierologici tipo-specifici per
l’infezione da HSV
Nome
Abbr.
Riferim. Bibliog.
Immunodot enzyme assay gG-IEA
Lee et al. 1985; Lee et al. 1986
Western blot
WBA
Ashley et al. 1988/1991
Immunoblot gG assay
gG-blot
Sanchez-Martinez et al. 1991a/1991b
Indirect gG-2 ELISA
gG-2 EIA
Ho et al. 1993
Monoclonal antibody
blocking RIA
MAb-RIA
Slomka et al. 1995
RIBATM Strip
Immunoblot Assay
RIBATM-SIA
Alexander et al. 1996; Ashley et al.
1997
Gull gG-EIA
Gull EIA
Ashley et al. 1997
MRL gG-EIA
MRL-EIA
Prince et al., 2000
HSV.ELISA IgG
MRL-ELISA
Palù et al., 2001
Tests to Use
ELISA. Few companies make and sell this test kit in the United States
and many countries around the world. Some of these tests have been
approved by the FDA for diagnosing HSV infection in adults, including
pregnant women. The test takes less than a day once the specimen
arrives in the laboratory.
Immunoblot. This test usually contains the same recombinant gG-1 and
gG-2 that is used in the ELISA kits. Some of them are also FDA approved
They detect antibodies to both HSV-1 and HSV-2 on a paper strip and are
more expensive than the ELISAs.
Immunodot. This test is a near-patient test that can be performed in
offices and clinics that have laboratory facilities. It is a membranebased immunoassay for qualitative determination of igG specific for
HSV-2. The test employs a semi-purified antibody-binding protein,
conjugated to colloidal gold particles and semi purified gG2. Only a
couple drops of blood from a finger-stick is needed. This test requires
about 10 minutes to perform and read.
ELISA
These are the packages from Focus Technologies’
tests. Note that the company has a label on the
packaging to indicate that the tests are gG Based.
This should help the health care provider and
his/her laboratory to know that they have
purchased one of the new type specific tests.
IMMUNOBLOT
These are actual immunoblot test strips
photographed in the divided container used to
perform the test. Each strip is a test for a different
patient. The arrows point to the gG-2 (HSV-2) and
gG-1 bands (HSV-1) on two of the strips. Most of
the tests in the run have HSV-1 antibodies.
Immunoblot IgG assay
(HerpeSelect™ 1 and 2)
Controllo interno
(siero umano)
Antigene comune
(HSV)
gG1
gG2
1&2
Pos
1
Pos
2
Pos
Eqv
Neg
Neg
Il test e’ considerato valido se:
1)Il controllo interno e’ positivo
2)Se la banda specifica per gG1 e/o gG2 + la banda relativa
all’antigene comune per HSV-1 e 2 sono visibili
Neg
Invalid
HerpeSelect™ 1 and 2
Immunoblot IgG
Single strip containing
 serum control
 common antigen
 recombinant gG1
 recombinant gG2
Test Highlights
 Single kit tests for both serotypes
 20 uL serum
 Approx. 2 3/4 hrs. for testing
 Minimal equipment (platform rocker)
 Best for lower volume testing
IMMUNODOT
The biokit HSV-2 test is fast and easy to perform. This photo
shows a positive patient result (right side of window) and control
dot (left side of window). Normally positives are less red than
the control dot, but can be as dark as the control dot. If HSV-2
antibodies are not present, only one red dot appears. Only one
red dot represents a "negative" result. If a patient has only HSV1 antibodies, the test will read "negative" with only one dot
being red because this test does not contain gG-1. If neither dot
turns red the test is invalid and must be repeated.
Western Blot
E’ il golden standard per la diagnosi
sierologica tipo specifica dell’infezione da
HSV:
These are Western blot tests from one patient who presented
with recurrent HSV-2 infection (Year 0). At 1.1, 1.9, 3.2, and
8.2 years later, serum was drawn and tested again. The lefthand blot in each pair has HSV-1 proteins and the right-hand
blot has HSV-2 proteins. Someone trained in Western blot will
recognize the gG-2 band and also see that the gG-1 band is
missing.
Interpreting the gG Serology (1)
Test results from one of the glycoprotein G specific tests
(ELISA or Immunoblot) are as follows:
HSV-2 Antibodies Present: This means the patient has HSV-2
infection, the virus may be latent in the sacral nerves
HSV-2 Antibodies NOT Present: This means with 95-98%
accuracy the patient does not have genital herpes unless he
acquired it very recently. New studies have shown that by 6
weeks after HSV-2 infection, 60-75% of patients will be
positive by ELISA (the HerpeSelect® ) test but for a few
patients (about one in 5), it can take as long as 6 months
to develop antibodies.
If the patient might have acquired herpes very recently, he
may need to repeat the antibody test in 6-8 weeks.
Interpreting the gG Serology (2)
HSV-1 Antibodies Present: This means the patient has
HSV-1 infection. The antibody assay cannot tell where
HSV-1 is latent. In most people (>90%) it is in the
nerves of the mouth and eyes. Some people do have
genital HSV-1 and could have HSV-1 antibodies from
genital HSV-1 infection.
Neither HSV-1 or HSV-2 Antibodies Present: The
patient is not infected with either HSV-1 or HSV-2 but
he is susceptible to getting infection. There is a small
chance that he may have been recently infected, but
has not made antibodies yet.
Potential population in whom HSV
type-specific serology could be
useful
To identify subclinical HSV-2 carriers
Candidates for behavioural intervention
Candidates for antiviral therapy
Potentially reduce transmission
To identify pregnant women at risk
Those who are uninfected but have serologically
discordant partners
Those already infected
Potential population in whom HSV
type-specific serology could be
useful
To identify asymptomatic individuals in other
groups
Semen donors
Patients entering immunosuppressive therapy
Patients with HIV infection
To identify candidates for vaccines
Suggested ordering guidelines
for HSV serological tests
Situation
Serologic
test(s)
Comments
Symptomatic,
virus positive
None
A virologic test is always better
Symptomatic,
negative
Serum pair
(non-typing)
Misleading in HSV-1+ with new
HSV-2 infection
Serum pair (typing)
Very helpful
Asymptomatic,
worried, > 8 weeks
post exposure
Non-typing
Helps only if seronegative
Typing
Positive test cannot correlate
infection to exposure event
High risk, history
negative
Non-typing
Helpful only if negative
Ricadute cliniche della diagnosi
di laboratorio
Epidemiologia
Prognosi
- HSV-1
- HSV-2
Valutazione del rischio di trasmissione
- herpes neonatale
- partner
- associazione con HIV e/o con altri patogeni STD
Valutazione
- Genotipica
- Fenotipica
SIFILIDE
SIFILIDE: EZIOLOGIA E STORIA
La sifilide, una malattia complessa sessualmente trasmissibile
causata dal batterio Treponema pallidum, fu descritta per la
prima volta nel XVI secolo e si ritiene che sia stata importata dalle
Americhe dopo i primi viaggi degli spagnoli.
Nei paesi industrializzati, l’incidenza della sifilide iniziò a calare
verso la fine del 1800, per poi avere un altro picco dopo la Prima
Guerra mondiale.
Dopo la Seconda Guerra, grazie anche alla disponibilità di metodi
diagnostici efficaci e al trattamento con antibiotici, la malattia
ebbe una nuova riduzione, anche se negli ultimi anni la sua
incidenza è andata aumentando sia nei paesi in via di sviluppo
che in alcuni paesi europei.
Dopo l’AIDS, la sifilide, che ha una incidenza annua di 12 milioni
di nuovi malati nel mondo, è la malattia sessualmente
trasmissibile con il più alto tasso di mortalità.
AGENTE EZIOLOGICO
IL Treponema Pallidum, appartiene alla famiglia delle
treponematacee, batteri di forma elicoidale, mobili, a divisione
trasversale.
La loro lunghezza variada 8 a 14 um e la sua larghezza da 0.15 a 0.20
um.
La coltura in vitro dei treponemi non è stata ancora realizzata. Per
tale motivo il loro metabolismo è poco conosciuto.
Nel mondo si registrano 12 milioni di nuovi casi di sifilide all’anno(OMS,1999)
Nord America
100.000
Europa
140.000
Nord Africa
370.000
Sud America
3 milioni
Europa-est ed Asia
100.000
Est Asiatico-Pacifico
240.000
Sud Africa
4 milioni
Sud-Sudest Asia
4 milioni
Australia e
N.Zelanda
10.000
Aspetti epidemiologici
Nel mondo, secondo l’Oms, la sifilide colpisce circa 12 milioni di
persone, con grande presenza sia in Africa che in Asia e in America
Latina. Il numero di nuovi casi per anno (dal 1995 al 1999)
espresso in milioni di persone è il seguente:
Nord America
Europa occidentale
Nord Africa e Medio Oriente
Europa Orientale e Asia Centrale
Africa Sub Sahariana
Asia Sud e Sud-est
Asia orientale e Pacifico
Australia e Nuova Zelanda
America latina e Caraibi
1995
1999
0.14
0.20
0.62
0.10
3.53
5.79
0.56
0.01
1.26
0.107
0.136
0.364
0.105
3.828
4.038
0.244
0.008
2.928
Cusini M, Ghislanzoni M, Bernardi C, Carminati G,
Zerboni R, Alessi E, Suligoi B. Syphilis outbreak
in Milan, Italy. Sex Transm Infect. 2004
Apr;80(2):154.
SIFILIDE: ASPETTI GENERALI
La sifilide è una malattia genitale che causa ulcere ed
escoriazioni e facilita la trasmissione dell’Aids (rischio di
trasmissione del virus HIV è da 2 a 5 volte più elevato)
Grazie a un semplice test diagnostico e a una elevata efficacia
della cura antibiotica, è oggi una malattia potenzialmente
controllabile dai sistemi di sanità pubblica.
Se non è trattata adeguatamente però può causare danni al
sistema nervoso, ai vasi arteriosi, disordine mentale ed
eventualmente, morte.
La sifilide si trasmette di persona in persona direttamente
attraverso le ferite e le ulcere che si formano nelle zone
genitali, rettali e sulla bocca a seguito di contatto sessuale.
La malattia può facilmente essere trasmessa
STADI DELLA MALATTIA (1)
La SIFILIDE si sviluppa in diversi stadi, ciascuno caratterizzato
da sintomi e decorso diverso.
Sifilide primaria
Tra l’infezione e l’insorgenza dei primi sintomi possono passare da 10 a 90
giorni (mediamente venti giorni). Questo stadio è caratterizzato dalla comparsa di
una singola ferita, o da più pustole. Normalmente la ferita è consistente, tonda,
piccola e indolore e compare nel punto in cui avviene l’infezione batterica. Questa
ferita dura 3-6 settimane e guarisce da sola. Se la malattia non è trattata in
questa fase, evolve verso uno stadio secondario.
Sifilide secondaria
Inizia quando si ha l’insorgenza di una eruzione cutanea in più punti, senza
prurito. Questa eruzione può comparire durante la fase di scomparsa della ferita, o
anche dopo settimane. L’eruzione è solitamente rossastra o bruna, con macchie sia
sui palmi delle mani e dei piedi o in altre parti del corpo. A volte le macchie sono
diverse e ricordano eruzioni tipiche di altre malattie. Anche senza alcun
trattamento, l’eruzione sparisce da sola. Tra i sintomi tipici di questo stadio
possono esserci anche febbre, linfonodi ingrossati, mal di gola, perdita di
capelli a chiazze, mal di testa, perdita di peso, dolori muscolari,
stanchezza.
STADI DELLA MALATTIA (2)
Sifilide avanzata (stato latente e terziaria)
Alla scomparsa dei sintomi del secondo stadio, la persona è ancora malata anche se
non mostra più i sintomi evidenti. In questa fase, possono iniziare i danni agli
organi interni, al cervello, ai nervi, agli occhi, al cuore e ai vasi sanguigni, al
fegato, alle ossa e alle articolazioni. I danni interni possono manifestarsi anche
anni dopo la comparsa dei primi sintomi. A questo punto la sifilide entra nel
terzo stadio, anche se danni neurologici possono manifestarsi già dal secondo stadio
(sifilide neurale). In questa fase l’individuo perde la capacità di controllare i
movimenti muscolari, può avere delle paralisi, confusione mentale, cecità
graduale e sviluppo di demenza. Il danno può essere tanto serio da portare
alla morte.
Sifilide congenita
A seconda dello stato d’infezione della madre, la malattia può essere trasmessa al
feto causando morte in utero (40 per cento dei casi) o la nascita di un bimbo
già infetto, con sifilide congenita (70 per cento dei casi).
Se la madre ha avuto la malattia nei quattro anni precedenti la gravidanza, il rischio
di trasmissione al feto è molto elevato. I sintomi possono anche essere assenti
al momento della nascita e comparire successivamente, causando se non
trattati adeguatamente anche serie complicazioni allo sviluppo del bambino.
GRAVIDANZA E SIFILIDE CONGENITA
La sifilide durante la gravidanza può determinare:
• Aborto
• Nascita di un feto morto
• Parto prematuro
• Morte neonatale
• Parto a termine con neonati con infezione clinicamente silente (da un terzo e
metà dei casi): spesso i neonati non presentano i segni e i sintomi della malattia,
che possono comparire dopo mesi o anni oppure rimanere silente per tutta la
vita
• Parto a termine con neonati con infezione clinicamente manifesta
EPIDEMIOLOGIA
L’aumento dell’incidenza di sifilide negli adulti si ripercuote
sull’infezione congenita.
Nel 1999 negli USA sono stati registrati 556 casi.
DIAGNOSI DI LABORATORIO
La diagnosi di sifilide può essere effettuata utilizzando un’analisi al
microscopio di materiali prelevati da una escoriazione o da una
ferita del paziente.
La diagnosi puo’ essere effettuata anche con test serologico
Esistono due tipi di test serologici:
NON Treponema-specifici, (Venereal Disease Research
Laboratory [VDRL] e Rapid Plasma Reagin [RPR])
Treponema-specifici (p.e. fluorescent treponemal
antibody absorbed [FTA-ABS] e T. pallidum particle agglutination
[TP-PA]).
Il livello di anticorpi rimane nel sangue per mesi e anni anche dopo
il trattamento completo della malattia.
Dati gli effetti della sifilide contratta prima o durante la gravidanza,
lo screening per la presenza di anticorpi anti-Treponema dovrebbe
essere effettuato assieme agli altri test serologici nelle prime
settimane di gestazione.
DIAGNOSI DI LABORATORIO
1. Diretta
Microscopia in campo oscuro
Immunofluorescenza specifica su vetrino (direct fluorescent
antibody Treponema pallidum [DFA-TP])
PCR
2.Sierologica
- Esami non treponemici
- Esami treponemici
DIAGNOSI DIRETTA
DARK-FIELD MICROSCOPY
Dark-field microscopy is the most specific technique for
diagnosing syphilis when an active chancre is present. However,
its accuracy is limited by the experience of the operator
performing the test, the number of live treponemes in the lesion,
and the presence of nonpathologic treponemes in oral or anal
lesions.
In preparation for dark-field microscopy, the lesion is cleansed
and then abraded gently with a gauze pad. Once a serous
exudate appears, it is collected on a glass slide and examined
under a microscope equipped with a dark-field condenser.
T. pallidum is identified by its characteristic corkscrew
appearance.
Given the inherent difficulties of dark-field microscopy, negative
examinations on three different days are necessary before a
lesion may be considered negative for T. pallidum.
PCR
The polymerase chain reaction (PCR) has been used to
detect Treponema pallidum infection.
T. pallidum has been found by PCR in the
blood (Pietravalle et al, 1999 and Marfin et al, 2001)
lymph nodes (Kouznetsov and Prinz, 2002),
rashes (Sutton et al, 2001)
stomach (Inagaki et al, 1996),
aortal wall (O’Regan et al, 2002),
or cerebrospinal fluid (Noordhoek et al, 1991)
A multiplex PCR assay which simultaneously detects T.
pallidum, HSV-1 and H. ducrey has been used to provide
an etiological diagnosis for patients with GUD as well as
for validating syndromic management algorithms
DIAGNOSI INDIRETTA
NONTREPONEMAL TESTS
Syphilitic infection leads to the production of nonspecific antibodies
that react to cardiolipin. This reaction is the basis of traditional
nontreponemal tests such as the VDRL (Venereal Disease Research Laboratory)
test and rapid plasma reagin test.
With nontreponemal tests, false-positive reactions can occur because
of pregnancy, autoimmune disorders, and infections. In addition, these
tests may show a "prozone" phenomenon in which large amounts of
antibody block the antibody-antigen reaction, causing a false-negative
test in the undiluted sample.
Qualitative nontreponemal tests are widely used for syphilis screening.
However, their usefulness is limited by decreased sensitivity in early
primary syphilis and during late syphilis, when up to one third of
untreated patients may be nonreactive.
After adequate treatment of syphilis, nontreponemal tests
eventually become nonreactive. However, even with sufficient
treatment, patients sometimes have a persistent low-level positive
nontreponemal test (referred to as a serofast reaction).
Titers are not interchangeable between different test types.
Hence, the same nontreponemal test should be used for follow-up
evaluations.
BASIS OF NON-TREPONEMAL TESTS
Capture system
Liposomes in suspension visible
flocculation with lipoidal antibodies
Liposomes in suspension + unattached
charcoal particles producing dark
coloured flocculation due to trapping
of charcoal particles in lattice
formed by Ag.Ab complex
VDRL antigen coated onto wells of
microtitre plates and attached
antibody detected by enzyme
immunoassay
VDRL antigen coated ono well of
microtitre plates; attached antibody
detected by anti-IgG plus anti-IgMcoated red blood cells
Test
VDRL
RPR
EIA (Reagin)
SPEA (solid phase erytrocyte
adherence)
NON TREPONEMAL TESTS
• Non Treponemal tests can be qualitative or semiquantitative
• RPR and VDRL titres are raised in patients with acute
infection, reinfection or reactivation
• About 72.84% of patients with primary or secondary syphilis
show a 4-fold decrease in their RPR or VDRL titer 6 months
after completing appropriate treatment
• The rate of seroconversion depends on the pretreatment titre
and stage of disease
• Non treponemal tests are useful not only in identifying active
infection but also in monitoring the effectiveness of treatment
VDRL
VDRL Antigen is a non treponemal preparation specially
developed for the rapid detection and semi-quantification by
coagulation on a slide of plasma reagins, a group of
antibodies detected against tissue components
produced by almost every patient infected with
Treponema pallidum.
The assay is performed by testing the antigen, an
association of 0,2% lecithin, 0,03% cardiolipin and 0,9%
cholesterol, against unknown samples. The presence or
absence of a visible flocculation or agglutination indicates
the presence or absence of circulating antibodies in the
samples tested.
The test permits a rapid screening of a large number of
samples so that reactors can be give immediate treatment.
In the particular case of blood banks the test allows the
quick identification of all serological reactive blood samples.
TREPONEMAL-SPECIFIC TESTS
Treponemal-specific tests detect antibodies to antigenic components
of T. pallidum.
These tests are used primarily to confirm the diagnosis of syphilis in
patients with a reactive nontreponemal test.
Treponemal-specific tests include the EIA for anti-treponemal
IgG, the T. pallidum hemagglutination (TPHA) test, the
microhemagglutination test with T. pallidum antigen, the
fluorescent treponemal antibody-absorption test (FTA-abs),
and the enzyme-linked immunosorbent assay.
Treponemal tests have sensitivities and specificities equal to or
higher than those for nontreponemal tests. However, treponemalspecific tests are more difficult and expensive to perform, which
limits their usefulness as screening tests.
False-positive results can occur, especially when the FTA-abs test
is used in patients with systemic lupus erythematosus or Lyme
disease.
Unlike nontreponemal tests, which show a decline in titers or become
nonreactive with effective treatment, treponemal-specific tests
usually remain reactive for life. Therefore, treponemal-specific
test titers are not useful for assessing treatment efficacy.
BASIS FOR TREPONEMAL TESTS
Antigen
Capture system
Test
Intact treponemes
Treponemes fixed onto
microscope slides
FTA-ABS
Purified and sonicated
treponemes
Attached to red blood
cells
TPHA
Attached to gelatin
particles
TPPA
Attached to microtiter
plates
EIA
Protein separated by
PAGE and transferred
to filters by WB
Immunoblot
Attached to microtiter
plates
Recombinant EIA
Attached to latex
particles
Latex agglutination
Recombinant antigens
TPHA AND FTA-ABS
Both the Treponema pallidum haemagglutination assay and the
fluorescent Treponema antibody tests are highly specific for
Treponema antigens:
•TPHA - sheep red blood cells coated with T. pallidum are
agglutinated by patient's antibody
•FTA-ABS - T. pallidum is fixed to a microscope slide, antibodies in
patient's serum attach and are detected by the addition of fluorescent
anti-human immunoglobulin.
•The test is positive in 90% of patients with primary infection
and positive in all patients with secondary or tertiary infection.
It can be adapted to detect either IgG or IgM antibody. This test
becomes positive in early disease (at about 3-4 weeks after infection at the same time as the reagin tests).
False positives may result from non-sexually transmitted treponemal
diseases of the tropics - such as Yaws and Pinta. Differentiation
requires a careful history and examination.
ICE Syphilis EIA
The ICE Syphilis EIA uses three recombinant
T. pallidum antigens (TpN15, TpN17, and TpN47)
coated onto the wells of microtiter plate strips
The wells are also coated with anti-human
immunoglobulin G (IgG) and IgM.
The antitreponemal component of the captured
antibodies is detected by peroxidase-conjugated
recombinant antigen (TpN15, TpN17, and TpN47)
EIA is ideally suited for the screening of large numbers
of specimens because it can be readily automated, the
results are read objectively, and reports may be
generated electronically, removing any risk of
transcriptional errors
New treponemal tests
• More than 20 companies manifacutre rapid, simple
treponemal tests that can use whole blood, serum or
plasma “http://www.rapid-diagnostics.org/files/SyphilisManufacturers-website.rtf”
• Most use immunochromatographic strips coated with
antigens of T. pallidum
• Antigen-antibody reactions appear as a coloured line or
spot on the membrane.
• Some use a format similar to RPR where latex particles
are coated with treponemal antigens
• Most of these rapid tets are appropriate for use in
primary health-care setting as they require minimal
training and give a read-ou in 8-20 min
• Evaluation are needed
Conclusioni (1)
In practice serolical tests for syphilis are used for:
• Screenin asympomatic individuals with no history
suggestive of syphilis, such as pregnant women;
• Screening genitourinary medicine clinic attenders
at recent risk of acquiring a STD infection
• Screening blood and organ/tissue donors;
• Detecting or excluding current or past syphilis in
patients with HIV infection
• Testing patients whose history or clinical signs are
consitent with syphilis
• Confirmatory testing of specimens reactive in screening
tests for syphilis
• Assesment of the stage of infection and monitoring the
therapeutic response
Conclusioni (2)
• The testing strategy employed varies
• Either a non treponemal test alone, a
treponemal test alone, or both in
combination may be used depending on
several factors, including whether the
ain is to detect all stages of shyphilis or
only infectious syphilis
Conclusioni (3)
Stage
Diagnosis (sensitivity)
Primary syphilis
Dark-field microscopy of skin lesion (80%)
Nontreponemal tests (78% to 86%)
Treponemal-specific tests (76% to 84%)
Secondary syphilis
Dark-field microscopy of skin lesion (80%)
Nontreponemal tests (100%)
Treponemal-specific tests (100%)
Latent syphilis
Nontreponemal tests (95% to 100%)
Treponemal-specific tests (97% to 100%)
Tertiary (late) syphilis
Nontreponemal tests (71% to 73%)
Treponemal-specific tests (94% to 96%)
Neurosyphilis
Cerebrospinal fluid examination
SCREENING
VDRL e TPHA sono i test di screening per eccellenza
I test non treponemici utilizzati in combinazione con quelli
treponemici hanno un valore predittivo alto e i risultati positivi
sono probabilmente indicativi di un’ infezione reale
DIAGNOSI DELLA SIFILIDE CONGENITA
Per stabilire se un neonato da madre portatrice dell’infezione
non adeguatamente trattata, sia effettivamente infetto le indicazioni
diagnostiche si basano:
• sulla ricerca delle IgM specifiche (fluorescent treponemal
antibody absorption test used with fractionated serum, FTA-ABS
19S IgM test).
• sulla evidenziazione diretta del microrganismo mediante PCR o
test di immunofluorescenza su siero e liquido
cefalorachidiano del neonato.
I neonati di madri sieroreattive devono essere trattati con
penicillina, indipendentemente dal fatto che la madre fosse stata o
meno trattata in gravidanza.
La sieronegatività al parto può non essere un indice
certo di non-infezione materna. Quindi, in caso di madre a
Rischio, bisogna ritestare la donna e il figlio, nei mesi successivi al
parto.
PREVENZIONE DELLA SIFILIDE CONGENITA
La prevenzione della sifilide congenita si attua
mediante il controllo sierologico in gravidanza e il
trattamento delle donne infette e dei loro partners.
La sifilide congenita si può evitare quindi
praticando i test sierologici, in quanto un adeguato
trattamento farmacologico effettuato alla madre
prima della fine del 4° mese di gravidanza
evita l’ infezione al feto.
Diagnosis IN SUMMURY
•Dark-field microscopy is important in evaluating moist cutaneous
lesions, such as the chancre of primary syphilis or the condyloma lata of
secondary syphilis. When dark-field microscopy is not available, direct
immunofluorescence staining of fixed smears (direct fluorescent antibody
Treponema pallidum [DFA-TP]) is an option. Both procedures detect the
causative organism at a rate of approximately 85-92%.
•In suspected acquired syphilis, perform nontreponemal serology
screening using Venereal Disease Research Laboratory (VDRL), rapid
plasma reagin (RPR), or the recently developed ICE Syphilis
recombinant antigen test. Then, test sera yielding a positive or
equivocal reaction by the fluorescent treponemal antibody-absorption
(FTA-ABS), quantitative VDRL/RPR, and microhemagglutination assay
Treponema pallidum (MHA-TP) tests.
•For evaluation of infants with suspected congenital syphilis, the 19S
immunoglobulin M FTA-ABS serology test or the Captia Syphilis-M test
currently is recommended. Every pregnant woman should undergo a
nontreponemal test at her first prenatal visit, and women at high
risk of exposure should have a repeat test in the third trimester and again
at delivery.
CONCLUSIONI
Il controllo della sifilide dipende dalla
combinazione di competenze:
1. Cliniche
2. Epidemiologiche
3. Laboratoristiche
CANCROIDE O ULCERA MOLLE
CHANCROID
1. INTRODUCTION
Chancroid is a sexually transmitted disease caused by infection with
the bacterium Haemophilus ducreyi. This organism causes one or
more ulcers on the genitalia and are associated with inguinal
lymphadenitis.
The affected lymph nodes may progress towards abscess formation.
Co-existent infection with other organisms e.g. Herpes simplex,
Treponema pallidum and Chlamydia trachomatis is common.
A diagnosis of chancroid is based on the typical clinical findings and
exclusion of other conditions. Strictly speaking, a definitive
diagnosis of chancroid should only be made where H. ducreyi is
recovered from genital ulcers.
2. EPIDEMIOLOGY
Chancroid is endemic in tropical and subtropical countries, but it
is sporadic in temperate countries.
The disease was prevalent in war-times.
Recently, there have been outbreaks in the United States,
Canada and some European countries.
It is thought that prostitutes constitute an important reservoir of
infection.
The disease is also more common in the uncircumcised men,
and in unhygienic and low socioeconomic conditions.
Asymptomatic carriers exist.
3. AETIOLOGY
The causative organism, Haemophilus ducreyi, was first
described by Ducrey in 1889.
It is a Gram negative coccoid-bacillary rod
predominantly located in the extra cellular spaces.
H. ducreyi is a fastidious organism, requiring stringent
conditions for a successful growth in vitro.
Chancroid is almost exclusively a sexually transmitted
disease. However, there have been reports that
susceptible medical personnel acquired extragenital
lesions by accidental inoculations
5. DIFFERENTIAL DIAGNOSIS
The differential diagnosis includes syphilitic chancre,
herpes genitalia, granuloma inguinale, secondary
pyogenic infection of traumatic lesion, excoriated scabies,
neoplasm and allergic conditions.
Syphilis and genital herpes must be differentiated from
chancroid.
One of these co-exists in about 10% of patients with
chancroid.
Typical chancroidal ulcers are undermined, invariably tender
and are deeper than herpetic ulcers. In chancre, the ulcer is
indurated and painless. LGV gives multilocular buboes but
ulcers are inconspicuous.
6. INVESTIGATIONS
A diagnosis of Chancroid can be made when the clinical
features are typical and other venereal causes of genital
ulcerations have been excluded.
Investigations like viral culture for Herpes simplex; dark ground
examination of a smear from the ulcer for T. pallidum and
serological tests for syphilis.
Gram-stained smear from the ulcer can also be performed in
suitable cases looking for the Gram negative coccobacillary rods
which form long trails within mucous strands giving a 'shoal of
fish' appearance.
Culture plates containing enriched GC media are useful for
cultivation of this fastidious organism, and can be available upon
arrangement with the laboratory.
Laboratory Studies (1)
Gram stain
The sensitivity of Gram stain to diagnose chancroid has been
reported to range from 10-90%.
The classic description of H ducreyi on Gram stain is that of a
"school of fish" with small, pleomorphic, gram-negative rods.
Most experts agree that Gram stain has limited utility in diagnosing
chancroid.
Culture
Definitive diagnosis requires culturing H ducreyi on special culture
medium that is not always readily available.
Even with the proper medium, the sensitivity is not higher than
80%.
Serology: Serologic testing has been hindered by the inability to
distinguish acute from past exposure.
Newer diagnostic techniques are evolving rapidly. DNA probes
and polymerase chain reaction (PCR) appear promising, with high
sensitivity and specificity.
Laboratory study (2)
On primary isolation media, growth may be visible at 24 hours but
identifiable colonies may not be seen until after 48 to 72 hours of
inoculation.
Culture plates should not be discarded as negative until after at
least five days of inoculation.
A Gram-stained smear should be performed on colonies suspected
of being H. ducreyi. Gram-negative bacilli compatible with H.
ducreyi should be biochemically tested.
In contrast to other haemophilus species, H. ducreyi requires
hemin (X factor) for growth and thus is positive in the porphyrin
test. H ducreyi does not require NAD (V factor) for growth.
The use of specific monoclonal antibodies to detect bacterial
antigens is sensitive, specific and less time consuming but are not
widely available.
Polymerase chain reaction (PCR) is now also employed to achieve
a higher diagnostic accuracy. Biopsy of the ulcer is rarely
performed.
LINFOGRANULOMA VENEREO
Background
Lymphogranuloma venereum (LGV) is a sexually
transmitted disease that primarily infects the lymphatics.
The disease originally was described in 1833 by Wallace. It
was defined as a clinical and pathological entity in 1913 by
Durand, Nicolas, and Favre.
LGV synonyms include lymphopathia venerea, tropical
bubo, climatic bubo, strumous bubo, poradenitis
inguinales, Durand-Nicolas-Favre disease, and
lymphogranuloma inguinale.
Pathophysiology
LGV is caused by Chlamydia trachomatis. It
gains entrance through skin breaks and
abrasions, or it crosses the epithelial cells of
mucous membranes. The organism travels via
the lymphatics to multiply within mononuclear
phagocytes in regional lymph nodes.
Transmission is predominantly sexual. However,
transmission by fomites, nonsexual personal
contact, and laboratory accidents has been
documented. The creation of aerosols of this
organism has been associated with infection and
pulmonary symptoms.
Frequency
•In the US: Sporadic cases occur in North
America, Europe, Australia, and most of Asia.
The majority of cases in the US involve
recent travel to an endemic area where the
patient was sexually active; therefore,
obtaining a travel history is important.
Historically, the average number of LGV
cases in the US has been fewer than 600 per
year.
•Internationally: LGV is endemic in East
and West Africa, India, Southeast Asia, South
America, and the Caribbean.
Causes
•The causal organism is C trachomatis,
serovars L1, L2, and L3.
•Serovar L2 is the most common cause.
•Risk factors
Unprotected sex
Anal intercourse
Residing in or visiting
tropical/developing countries
Prostitution
DIAGNOSI
Laboratory findings
•Initial laboratory analysis may reveal mild leukocytosis.
•These nonspecific results do not aid the clinician in the
diagnosis of LGV.
•Previously, the Frei test was the only method available to
identify a chlamydial infection. Currently, the Frei
intradermal test is only of historical interest.
•The test was based on a positive hypersensitivity to an
intradermal standardized antigen, lymphogranuloma
venereum, which indicated past or present chlamydial
infection. The Frei test would become positive 2-8 weeks
after infection.
•Unfortunately, the Frei antigen is common to all
chlamydial species and is not specific to LGV. Commercial
manufacturing of Frei antigen was discontinued in 1974.
Laboratory findings
•Complement fixation (CF) is more sensitive than the
Frei skin test, but it has some cross-reactivity with other
chlamydial species. CF sensitivity is 80% for LGV.
•A test titer of 1:16 is strongly suggestive of LGV and a
titer of > 1:64 indicates active LGV. A 4-fold rise or fall
in titer further supports the diagnosis.
•The microimmunofluorescence test for the L-type
serovar of C trachomatis is the most sensitive and
specific test. Availability of this test is the limiting factor.
•Dermatopathology is not pathognomonic for LGV, and
cytology using Giemsa stain or iodine stain fails to
provide a high percentage of diagnoses.
•Definitive diagnosis may be made by aspiration
of the bubo and growth of the aspirated material
in cell culture. C trachomatis can be cultured in as
many as 30% of cases.
Granuloma inguinale (Donovanosi)
Granuloma inguinale
Background: Granuloma inguinale (GI) is primarily a sexually
transmitted disease in which characteristic intracellular
inclusions called Donovan bodies may be seen. It usually
manifests as genital lesions, which are indolent, progressive,
ulcerative, and granulomatous.
Pathophysiology: GI is caused by Calymmatobacterium
granulomatis, a gram-negative pleomorphic bacillus. The mode
of transmission is primarily through sexual contact, although GI
may be obtained through a fecal route or by passage through
an infected birth canal. It is considered to be only mildly
contagious, and repeated exposure may be necessary for
clinical infection to occur.
Frequency
•In the US: Fewer than 100 cases are reported
annually, many of which are thought to be due to
foreign travel.
•Internationally: GI is endemic in Western New
Guinea, the Caribbean, Southern India, South
Africa, Southeast Asia, Australia, and Brazil.
DIAGNOSIS
a) Calymmatobacterium granulomatis can not be
cultured.
b) The diagnosis is made by the identification of
intracellular “Donovan bodies” on a biopsy or smear
of lesion exudate using a Wright’s stain. Lesions are
slowly destructive and granulomatous.
CONCLUSIONI
ESAMI DI PRIMO LIVELLO: UN RIASSUNTO
L’OMS ha avviato, in collaborazione con la Banca Mondiale e l’Undp, un
programma speciale, la “Sexually Transmitted Diseases
Diagnostics Initiative (SDI)” con lo scopo di valutare i test
disponibili e mettere a punto linee guida per una diagnostica rapida
ed efficace da effettuarsi anche nei paesi poveri che hanno limitate
risorse sanitarie. L’iniziativa ha prodotto la pubblicazione di un
Manuale Operativo di test rapidi di laboratorio da effettuarsi nei diversi
casi (Laboratory-based Evaluation of Rapid Syphilis Diagnostics
Manual of Operations).
The tests to be included in this evaluation should have the following
operational characteristics:
1. Rapid -- test result is available in less than 15 min.
2. Simple-- test can be performed in a single or 2 steps, requiring
minimal training and no equipment
3. Easy to interpret --card or strip format with visual readout
http://www.who.int/std_diagnostics/publications/manuals/syphilis_eval
uation.pdf
ESAMI DI PRIMO LIVELLO:
UN RIASSUNTO
HSV
alcuni tests sierologici tipospecifici (biokit), MA con
prudenza
SIFILIDE
non treponemal tests
new treponemal tests
LGV
Test di Frei
ESAMI DI SECONDO
LIVELLO: UN RIASSUNTO
ESSENZIALI IN OGNI CASO PER
RAGGOCLIERE IL MAGGIOR NUMERO
POSSIBILE DI INFORMAZIONI E
PROCEDERE AL CORRETTO
MANAGEMENT DEL PAZIENTE CON
ULCERE GENITALI