Laboratory Diagnosis of Bacterial Sexually Transmitted Diseases

Laboratory Diagnosis of
Bacterial Sexually Transmitted
Diseases
Seminar on Infectious Diseases
- Diagnosing Common Infections in General Practice
5 July 2008
Kai Man KAM,
Consultant Medical Microbiologist,
Public Health Laboratory Centre,
Centre for Health Protection,
Department of Health, Hong Kong.

Syndromic approach with utilization
of laboratory diagnostic tools
Extracted from Canadian Guidelines on STI , 2006 : Management and Treatment of Specific
Syndromes Syndromic Management of Sexually Transmitted Infections
2

Tests in Laboratory
Detection
Neisseria gonorrhoeae
Chlamydia trachomatis
Treponema pallidum
Ureaplasmas
Trichomonas vaginalis
Other pathogens from genital tract specimens
Antimicrobial
susceptibility test
Neisseria gonorrhoeae
3

Neisseria gonorrhoeae
Presumptive identification
– Morphology
0.5 – 1 mm in diameter after 48 hrs incubation
Isolated colonies: up to 3 mm in diameter
Greyish color
Opaque, raised and glistening
– Oxidase test
Oxidase positive
– Gram stain
Gram negative diplococci

Neisseria gonorrhoeae
Confirmatory identification
– CTA sugar test
Determines acid production from carbohydrates in cystine
trypticase agar, phenol red as indicator
Yellow: +ve+
; Red/Orange:-ve
– API NH/Vitek
NHI/Rapid NH/Gonogen
II or 16S rRNA
PCR
Used when
– Isolates suspected of being gonococci fail to produce acid
from glucose
– Positive from extragenitial sites in patients at low risk for
gonorrhoeae
– Potential medico-legal implication
– Any doubt about the identity of the isolate

Interpretation
Interpretation
API NH/
API NH/Vitek
Vitek
NHI/Rapid
NHI/Rapid
NH/
NH/Gonogen
Gonogen II
II
Further
Further
confirmatory
confirmatory
tests
tests
performed
performed
-
-
-
-
N.
N. lactamica
lactamica
-
+
+
+
N.
N. meningitidis
meningitidis
-
-
+
+
N.
N.
gonorrhoeae
gonorrhoeae
-
-
-
+
ID
ID
Sucrose
Sucrose
Lactose
Lactose
Maltose
Maltose
Glucose
Glucose

Identification of N. gonorrhoeae
Growth on MTM
Greyish glistening colonies
Gram stain
+ve
Oxidase test
-ve
Not Neisseria spp.
Gram –ve diplococci
Others
Plate out on Columbia blood
agar for CTA sugar test &ST
Not Neisseria spp

Sensitivity Test for N.
gonorrhoeae
•Agar dilution Method
•Breakpoint plate MIC method
•Penicillin
•Ofloxacin
•Tetracycline
•Spectinomycin
•Ceftriaxone

Sensitivity Test for N. gonorrhoeae
•Disk diffusion Method
•Penicillin
•Ofloxacin
•Tetracycline
•Spectinomycin
• β - lactamase test

Interpretation
Zone Size (edge to edge) and Equivalent MIC Breakpoint for Neisseria gonorrhoeae
Antimicrobial
Agent
Disk
Content
Zone Size in mm (edge to edge,
D)
Equivalent MIC Breakpoint
(µg/ml)
Resistan
t
MR/LS
Sensitive
Resistan
t
MR/LS
Sensitive
Penicillin
10 units
≤10
10.5– 20
≥20.5
≥2
0.12–1
≤0.06
Ofloxacin
5µg
≤9
9.5—12
≥12.5
≥2
0.5–1
≤0.25
Tetracycline
30 µg
≤12
12.5–15.5
15.5
≥16
≥2
0.5–1
≤0.25
Spectinomyci
n
100 µg
≤4
4.5–5.5
5.5
≥6
≥128
64
≤32
Ceftriaxone
30 µg
—
—
≥14.5
—
—
≤0.25

Diagnostic Tests for
Syphilis
KM Kam,
Microbiology Division,
Public Health Laboratory Services Branch,
Centre for Health Protection, Dept. of
Health,

Syphilis Laboratory, PHLSB
Routine syphilis testing from
– Social Hygiene Service
– Clinical Service include HA,GOPC
– Antenatal include MCHC, HA
Reference laboratory service of diagnostic tests
Syphilis serology laboratory Quality Assurance
Program provider
Operational Research
12

Laboratory diagnostics
Detection of bacterium
– Animal inoculation (not routinely done )
– Dark-field microscopy
– Molecular detection
Serology : non-treponemal
& treponemal
13

Dark Field
• Requires darkfield microscope
• Cannot be used for oral or rectal lesions
(Because spirochetes are part of the normal flora)
• High complexity
• Easy to confuse fibers and Brownian
motion for spirochetes, if inexperienced
14

Dark Field
Advantages
Disadvantages
• Can be done while patient
waits
• Requires living,
motile treponemes
• Relatively inexpensive
• Requires large
number of organisms to
be present
• Not specific
15

Serological Tests
Syphilitic infection provokes
Two types of antibodies in the host
anti-lipid
reagin antibody - measured by non
treponemal tests , VDRL/RPR
specific anti-treponemal
treponemal antibody – measured by
EIA, TPHA, TPPA, FTA, WB
16

Nontreponemal Tests
ANTIGEN :
FUNCTION:
USES:
Cardiolipin, , Lecithin, Cholesterol
Detect IgG & IgM antibodies released
from damaged host tissue or
treponeme cell surface.
- Screen for syphilis
- follow efficacy of treatment
- detect reinfection
- distinguish new from old infection
- diagnosis of neurosyphilis (CSF) test.
17

Nontreponemal Tests
Advantages
Disadvantages
• Inexpensive
• Can be titered to follow
treatment efficacy
• Frequently seroreverts
following adequate
treatment
• Chronic BFP indicator
of autoimmune disease
• High rates of false
positives in some
populations
• Prozone reactions
• Affected by
temperature
• May be nonreactive
in late infection
18

Rapid Plasma Reagin (RPR) 18-mm Circle Card Test
• Macroscopic test
• Can be used with heated or unheated serum
• Can be used with plasma
• It is the most widely used nontrponemal test
• The stabilized emulsion has an expiration date of 2
years
19

VDRL-CSF Test
• Requires a sensitized VDRL antigen suspension
- VDRL emulsion diluted 1 + 1 in 10% saline
(Because low antibody content on CSF samples diluted antigen
allows for agglutination)
• Can perform Qualitative and Quantitative tests
• A reactive test is indicative of past or present infection of
the central nervous sysytem
• The only test approved for diagnosing neurosyphilis
20

Treponemal Tests
ANTIGEN:
Entire treponeme, , either whole, disrupted
or cloned antigens
FUNCTION: 1) Detect T. pallidum-specific antibodies
2) Can differentiate between IgG and IgM
USES:
1) Confirmation of screening tests
2) EIA or TPPA as screening tests
3) Confirming physical signs of disease
21

Types of Treponemal Tests
• Fluorescent Antibody
- FTA-ABS
ABS
• Hemagglutination
- TPHA
• Agglutination
- TP-PA
PA
• Enzyme Immunoassays
• Western Blot / Line Immunoassay
22

Fluorescent Treponemal Antibody-Absorption
Absorption
(FTA-ABS)
ABS)
• Utilizes whole treponeme
• Requires fluorescent microscope with darkfield
capabilities
• Requires fluorescein isothiocyanate (FITC) labeled anti
human globulin
• Needs to be absorbed with Reiter protein to remove non-
specificity
• In some cases Lupus patients give a non specific reaction,
serum is then absorbed with calf thymus DNA
• It is a time consuming test
23

Microhemagglutination Test for T. pallidum
Antibodies (TPHA)
TPHA is a treponemal test for the serological detection of antibodies
to various species and subspecies of pathogenic Treponema , the
causative agents of syphilis, yaws, pinta, , and endemic syphilis.
A passive hemaglutination procedure based on the agglutination of
erythrocytes sensitized with T. pallidum antigen by antibodies found in
the patient’s s serum
Patient’s s serum is diluted in absorbing diluent containing sheep or
bovine red cells, sonicated Reiter treponeme, , and normal rabbit serum
24

T. Pallidum Passive Particle Agglutination Test (TP-PA)
PA)
The test is a passive agglutination procedure based on the
agglutination of gel particles sensitized with sonicated T. pallidum
antigen that has been partially purified
• Test is more sensitive than MHA-TP in primary
syphilis
• No heterophile cross reaction interference
• Slightly less specific than MHA-TP
25

Enzyme Immunoassay
Assay based on microtitration wells coated with T. pallidum
antigens. After proper incubation period of test specimens
containing specific antibodies, unbound antibodies are washed
away.
Specifically-bound
IgG or IgM reacts with a conjugated
horseradish peroxidase (HRP) monoclonal antibody during a
second incubation period.
Specifically-bound enzyme conjugate is detected by reaction
with TMB (tetramethylbenzidine) subtrate solution.
Enzymatic reaction is stopped using 1N sulphuric acid. Assay
measured spectro-photometrically
photometrically to indicate presence or
absence of IgG or IgM treponemal antibodies.
26

Current Syphilis Laboratory Testing Trends
For economic reasons, the current trend is to screen with a
treponemal EIA test and to confirm (with other treponemal
test) and a nontreponemal test.
This is done because a large number of sera can be tested.
It is less labor intensive and the results are objective
This trend is contrary to the previous screening practice
before the introduction of EIA because the emphasis is on
identifying active cases with nontreponemal tests such as
RPR or VDRL
In areas of low incidence, , the Positive Predictive Value
(PPV) is low and an increase in number of patients will be
those of treated cases or false positives
27

Syphilis algorithm in
Public Health Laboratory Centre
EIA Screening
Positive
Negative
Nontreponemal Test
VD/RPR: titre
Treponemal Test
TPPA
FTA
WB*
* WB is in pilot service with social hygiene service only
28

Effectiveness of EIA screening
strategy in antenatal group
VD pos
VD neg
Total
Yes
17
33
50
sensitivity : 34%
ppv : 89.5%
Sampling by test result
Routine 4595 consecutive sera from antenatal clinics
Syphilis
No
2
4543
4545
Total
19
4576
4595
spec : 99.9%
npv : 99.3%
EIA (P)
EIA (N)
Total
Yes
50
0
50
sensitivity:100%
ppv : 80.6%
Syphilis
No
12
4533
4545
Total
62
4533
4595
spec : 99.7%
npv : 100%
29

Diagnosis of Neurosyphilis
The only recognized test for diagnosis is VDRL – CSF
Only 50% sensitive
FTA-ABS ABS can be used to rule out Neurosyphilis
Reactive result can occur in other stages of syphilis
(Neurologic
involvement can occur in any stage of syphilis)
False positive can occur if contaminated with serum
30

Sensitivity of tests for Diagnosis of Neurosyphilis
Test
Sensitivity
Specificity
VDRL 50% 99%
RPR 40% 85%
TRUST 40% 85%
MHA-TP
65-86%
90%
FTA-ABS
ABS 96-100%
93%
CDC unpublished data
31

Detection by Molecular Technique
Two sets of primers were designed based
on two unique features of the DNA
polymerase I gene (polA)
– A high cysteine content (24(
cysteines)
– Additional insertions in the gene
32

Subtyping by Molecular Technique
A combination of the two typing schemes
were used to obtain maximal
discrimination.
– the first scheme was based on the size
(number) of the repeat of the arp gene
– the second scheme used the Mse I RFLP of
the tpr gene, the unique banding patterns
were designated with a lowercase letter
33

Subtyping by Molecular Technique
Number of repeat (arp(
gene)
Mse I RFLP (tpr
gene)
Pilley et al
Number of specimens
10
b
d
10 b
10 d
1
2
11
d
11 d
1
12
a
12 a
3
g
12 g
1
13
d
13 d
1
14
a
14 a
4
b
14 b
2
c
14 c
1
d
f
14 d
14 f
15
3
15
d
15 d
6
16
d
16 d
3
e
16 e
1
f
16 f
1
20
b
20 b
Source: Molecular Subtyping of Treponema pallidum Subspecies pallidum. Pillay etal
1
34

Bacterial vaginosis (BV)
Characterized by a shift in the vaginal flora from the
dominant flora of Lactobacillus species to mixed
vaginal flora including Gardnerella vaginalis,
Bacteroides species, Mobiluncus species, and
Mycoplasma hominis.
Presence of clue cells, ≥ 20% of epithelial cells, is
considered as significant.
If clue cells are

Pathogens
Definite Pathogens to be reported:
N. gonorrhoeae, S. pyogenes, T. vaginalis
Potential Pathogens to be reported: (depends on clinical
history, sites and/or growth)
H. influenzae, Anaerobes, Candida species, Group B,
C, G Streptococci, Enterobacteriaceae, S. aureus, P.
aeruginosa, Actinomyces species

Trichomonas vaginalis
a parasitic protozoan that causes trichomoniasis
which is the most prevalent non-viral STD in the
world.
Examination for Trichomonas
After 2 days’ incubation of Feinberg medium,
prepare a wet mount and examine under
microscope
“Gold standard method”
37

Other Detection Methods
Monoclonal-antibody
antibody-based based enzyme-linked
immunosorbent (EIA) assay
Molecular methods
– Probe hybridization, PCR and Real-time PCR
assays
– Detect various regions or genes of the genome
including
2.3 kb T. vaginalis fragment
the feredoxin gene
beta tubuline gene
highly repeated DNA sequences
18S ribosomal gene.
38

Molecular Methods
Probe Hybridization
– A commercial kit, Affirm VP System
Uses synthetic DNA probes to directly detect Gardnerella vaginalis
and Trichomonas vaginalis from a single vaginal swab
But, false negative results were encountered
– A dot-blot hybridization: target a 2.3 kb T. vaginalis DNA
fragment
PCR methods
– Several PCR assays developed: Beta-tubuline
tubuline gene, 2000 bp
repeated fragment, 18S rRNA gene
– Sensitivity and specificity: 97% - 100%
– two Real-time PCR developed: TaqMan and FRET-based
No post-PCR processing, decrease turn-around time and increase
throughput
Sensitivity: 86% - 93%; Specificity: 89% - 95%
39

Ureaplasma and Mycoplasma
Detection
Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma
parvum and Ureaplasma urealyticum are the etiological
microorganisms constituting of NGU, postpartum fever, infertility,
and pelvic inflammatory.
Many methods were developed for detecting mycoplasmas and
ureaplasmas :
– Culture (difficult in culturing various Mycoplasma and Ureaplasma
spp.)
– Antigen detection (difficult due to lack of humoral immune response
in most patients)
– DNA probes (16S rRNA gene)
– PCR (can be multiplexed to detect M. hominis, , M. genitalium, , U.
parvum & U. urealyticum)
Methods used in our laboratory:
– PCR
– Commercial Kit
40

International Quality Assurance
Programmes
Quality Assurance Programmes
– Treponema pallidum- CDC, USA
– Chlamydia trachomatis
Quality Control for Molecular Diagnostics (QCMD/ EU) programme
RCPA Quality Assurance Programme (Australia)
– Genital pathogens
RCPA Quality Assurance Programme
Gonococcal Antimicrobial Susceptibility Programme (GASP) for
WHO Western Pacific Region.