Why STI Diagnosis is Important

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Why STI Diagnosis is Important

• 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.

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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
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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 granul
omatis
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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, Behcets syndrome, fixed
drug eruption, Reiters 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).
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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.
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EPIDEMIOLOGY (2)
2. Globally, the most frequent cause of
STD-related 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.
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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
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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-Oct23(5)429-40. 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
Mar30(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 10-year prospective study.
Infect Dis. 2005 Feb 1191(3)333-8.
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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
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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.
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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
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DIAGNOSTIC APPROACH (3)

• C. Laboratory testing
• 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.

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..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.
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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
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Diagnosi di laboratorio
-Diagnosi diretta -Diagnosi indiretta -importa
nte per HSV -fondamentale per T.pallidum
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DIAGNOSI DIRETTA INFORMAZIONI GENERALI
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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.
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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.
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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.
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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.
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Herpes genitale
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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
Anterograde transport of virus to mucosal and
cutaneous genital tissues
Uptake of HSV by sensory nerves with retrograde
transport to sensory ganglia
Productive viral replication in sensory neurons
Host Immune Response Circulating and
Mucosal Antibody production Cytokine
production Cell-mediated immunity
Dorsal root ganglia
Establishment of non-replicating, latent
infection in sensory neurons
Limits viral replication at all sites
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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.  
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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.
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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
gondii-specific 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.
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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.
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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
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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.
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E IN ITALIA?
- Suligoi et al. Sex. Trasm. Inf., 2000 -Cusini
M et al. Sex. Trasm. Dis., 2000
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Sieroprevalenza per HSV-1 e HSV-2 in tre città
italianeStudio di sieroprevalenza HSV,
2000-012.014 soggetti della popolazione generali
di Genova, Roma e Lecce
HSV-1
HSV-2
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positivi
9
positivi
negativi
negativi
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Sieroprevalenza dellHSV-2 in adulti
Italia (987 soggetti di etàgt 20 anni della
popolazione generale) Studio IHF
Sieroprevalenza HSV-1 e HSV-2, 2000-01
15,0
5,5
9,9
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HSV-2 seroprevalence by age and gender in a MST
clinic, Milan
Study on 919 patients, 661 males and 258 females
HSV-2
Age in years
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• 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

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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.
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Manifestations of disease among individuals
infected with HSV-2
20 Recognized symptomatic
20 Asymptomatic
60 Unrecognized symptomatic
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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
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• DIAGNOSI DIRETTA
• Messa in evidenza di HSV -1 e -2 o di parti di
  questo (antigeni o acidi nucleici)

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Test di Laboratorio per la Diagnosi Diretta
SENSIBILITA/SPECIFICITA Alta (gt90)
Alta Bassa Alta Molto
elev Molto elev
TEST Isolamento virale (esame colturale) Rilevazi
one di Antigeni (immunofluorescenza su vetrino,
EIA) Rilevazione di Acidi Nucleici
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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
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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.
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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.
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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.
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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
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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.
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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

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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)

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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

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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.
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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.
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• 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."

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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.
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Metodi sierologici tipo-specifici per linfezione
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-Mart
inez 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
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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 membrane-based 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.
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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.
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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.
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• Immunoblot IgG assay
• (HerpeSelect 1 and 2)

Controllo interno (siero umano)
Antigene comune (HSV)
gG1
gG2
1 2
1
2
Eqv
Neg
Neg
Neg
Invalid
Pos
Pos
Pos
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 allantigene
comune per HSV-1 e 2 sono visibili
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• 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

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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 HSV-1 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.
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Western Blot
E il golden standard per la diagnosi sierologica
tipo specifica dellinfezione da HSV
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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 left-hand 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.
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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.
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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 (gt90)
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.
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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
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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
84
Suggested ordering guidelines for HSV serological
tests
Situation Serologic Comments
test(s) Symptomatic, None A virologic
test is always better virus positive Symptomatic,
Serum pair Misleading in HSV-1 with new
negative (non-typing) HSV-2 infection
Serum pair (typing) Very
helpful Asymptomatic, Non-typing Helps only if
seronegative worried, gt 8 weeks

post
exposure Typing Positive test cannot correlate
infection to exposure event High risk,
history Non-typing Helpful only if negative
negative
85
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
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SIFILIDE
91
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, lincidenza 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 lAIDS, 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.
92
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.
93
Nel mondo si registrano 12 milioni di nuovi
casi di sifilide allanno(OMS,1999)
94
Aspetti epidemiologiciNel mondo, secondo lOms,
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  1995              
  1999   Nord America 0.14                
0.107 Europa occidentale 0.20                
0.136 Nord Africa e Medio Oriente  0.62         
        0.364 Europa Orientale e Asia
Centrale 0.10                 0.105 Africa Sub
Sahariana 3.53                 3.828 Asia Sud
e Sud-est 5.79                 4.038 Asia
orientale e Pacifico 0.56         
       0.244 Australia e Nuova Zelanda 0.01    
             0.008 America latina e
Caraibi 1.26                 2.928
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                        Cusini M, Ghislanzoni
M, Bernardi C, Carminati G, Zerboni R, Alessi E,
Suligoi B. Syphilis outbreak in Milan, Italy. Sex
Transm Infect. 2004 Apr80(2)154.
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SIFILIDE ASPETTI GENERALI
La sifilide è una malattia genitale che causa
ulcere ed escoriazioni e facilita la trasmissione
dellAids (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
99
STADI DELLA MALATTIA (1)
La SIFILIDE si sviluppa in diversi stadi,
ciascuno caratterizzato da sintomi e decorso
diverso. Sifilide primariaTra linfezione e
linsorgenza 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 linfezione
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 secondariaInizia quando si
ha linsorgenza di una eruzione cutanea in più
punti, senza prurito. Questa eruzione può
comparire durante la fase di scomparsa della
ferita, o anche dopo settimane. Leruzione 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, leruzione
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. 
100
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 lindividuo
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 congenitaA seconda dello stato
dinfezione 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.
101
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

102
EPIDEMIOLOGIA
Laumento dellincidenza di sifilide negli adulti
si ripercuote sullinfezione congenita. Nel 1999
negli USA sono stati registrati 556 casi.
103
DIAGNOSI DI LABORATORIO
La diagnosi di sifilide può essere effettuata
utilizzando unanalisi 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.
104
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

105
DIAGNOSI DIRETTA
106
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.
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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 (ORegan 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
109
DIAGNOSI INDIRETTA
110
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.
111
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-IgM-coated red blood cells

• Test
• VDRL
• RPR
• EIA (Reagin)
• SPEA (solid phase erytrocyte adherence)

112
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

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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.
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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-spe
cific 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, treponemal-specific 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.
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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
Recombinant antigens Attached to microtiter plates Recombinant EIA
Attached to latex particles Latex agglutination
116
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.

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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
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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/
  Syphilis-Manufacturers-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

119
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

120
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

121
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
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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
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DIAGNOSI DELLA SIFILIDE CONGENITA

• Per stabilire se un neonato da madre portatrice
  dellinfezione
• 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.

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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.
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127

• 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.

128
CONCLUSIONI

• Il controllo della sifilide dipende dalla
  combinazione di competenze
• Cliniche
• Epidemiologiche
• Laboratoristiche

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CANCROIDE O ULCERA MOLLE
131

• CHANCROID
• 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.

132
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.
133
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
134
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.  
135
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