Download Diagnostic testing for STBBIs in 2012: What you should

Jared Bullard MD FRCPC
Paediatric Infectious Diseases & Medical Microbiology
Associate Medical Director
Cadham Provincial Laboratory
WRHA STBBI Conference
March 14, 2012

No conflicts of interest to declare
1.
2.
3.
4.
Provide a general overview of diagnostic
microbiology testing
Review the evolution of resistance in
Neisseria gonorrhea and its impact on
testing
Compare the traditional and reverse
screening algorithms for syphilis
Discuss HIV testing, in particular the role
and challenges of HIV POCT
1.
2.
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8.
Chase children
Bath and feed children
Read MSc papers about lymphoma
Associate Medical Director at Cadham
Provincial Laboratory
Pediatric Infectious Diseases consultant
Eat
Chase children
Sleep?
Host-based testing
Pathogen-based
testing

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Antibody generated to pathogen
Two main classes used in diagnosis:
◦ IgM (typically seen between 7-10 days postexposure)
◦ IgG (typically rises between 3-6 weeks)


IgM used to determine acute infection
IgG used to determine immune status BUT
also can indicate infection if 4-fold rise in
titre

False positives and false negatives:
◦
◦
◦
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
Immunocompromised
Impact of early treatment
Cross-reaction with similar pathogens
Infection or immunization?
Prolonged time to diagnosis

Culture and sensitivity (C/S):
Most basic component of microbiology lab
Grow whole organism
Can determine antibiotic susceptibility (AST)
Requires optimal specimens for maximum
diagnostic yield
◦ Takes time for results
◦ Can be compromised by antibiotic treatment
◦
◦
◦
◦

Minimum inhibitory concentration (MIC)
◦ The concentration of antibiotic that visibly inhibits
growth of the test organism
◦ Usually measured in µg/mL

Breakpoints
◦ Laboratory and clinical data supporting successful
treatment of an organism based on MICs
◦ Defined by groups such as Clinical Laboratory
Standards Institute (CLSI)



Susceptible (S) = MICs of the organism are
achievable in serum with typical antibiotic
doses; treatment with antibiotic should work
Indeterminate (I)= cure may be possible but
higher doses may be necessary
Resistant (R)= MICs are above achievable
serum concentrations of antibiotics; clinical
failure
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
Another type of serological test
Looking for pathogen-specific proteins
Directly on specimens
Will not distinguish viability of organism



Polymerase chain
reaction (PCR)
Nucleic acid
amplification tests
(NAATs)
Nucleic acid
sequence based
amplification
(NASBA)

Advantages:
◦ Very sensitive and specific
◦ Rapid turnaround time (TAT)
◦ Large volume testing

Disadvantages:
◦
◦
◦
◦
Higher costs
Limited to only what you look for
Contamination
Does not distinguish “living” from “dead” pathogen
Sensitivity:
 The probability that a positive test reflects
disease
Specificity:
 The probability that a negative test
represents no disease

Has been around a long time
◦ Described in Leviticus 15:1-3 “when any man has a
bodily discharge, the discharge is unclean”
◦ Also discussed in ancient Chinese medical writings
◦ Name of disease gonorrhea given by Galen in 2nd
century meaning “flow of seed”
◦ First described by Neisser in 1879, not cultured
until 1882




Gram negative
diplococci
Very sensitive to
drying, temperature
variation and fatty
acids
Fastidious, easily
“outgrown”
Media such as
modified ThayerMartin best for
isolation

Orophayrnx
◦ posterior pharynx and tonsillar crypts

Urethra
◦ no urination for 2 hours, rotate, milk

Cervix
◦ Endocervix

Rectum
◦ If fecal contamination, discard


Vagina
Urine
◦ Ideally no urination 2 hour pre-test; first 10-20 mL



Use dacron swab
Transport at room temperature (4° C
inhibitory)
Ideally have in lab for culture within 24 hours

In 2011:
◦ 107,443 urine specimens for NAAT tested
◦ 1124 positive (1.0%)
◦ Highest rates of testing and positivity in ages 1524 years

Contrast to number of isolates by culture
analyzed at CPL:
◦ 213 isolates referred between 2007-2010
◦ 385,356 urine NAATs over same time
Urine NAATs performed for GC at CPL 2000
to 2011
Number of urine NAATs
120,000
100,000
80,000
POS
60,000
NEG
Total
40,000
% Positive
20,000
0
2000
2001
2002
2003
2004
2005
2006
2007
Year of testing
2008
2009
2010
2011

18 volunteer Russian female university
students:
◦ Asked to rate and describe armpit sweat of 34
samples from healthy men, GC treated and GC
infected
◦ Rated sweat odour of men with GC infection as less
pleasant and more “putrid” (p = 0.027) then health
and treated men who tended to be more “floral” (p
= 0.004)
1. Moshkin et al. Journal of Sexual Medicine, epub December 2011.
Preferred regimen:
 Cefixime 400 mg PO x 1 dose
Alternative regimens:
 Ceftriaxone 125 mg IM x 1 dose
 Azithromycin 2 g PO x 1 dose
 Ciprofloxacin 500 mg PO x 1 dose
 Spectinomycin 2 g IM x 1 dose
1.
PHAC, Canadian STI Guidelines, 2010
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Initially treated with sulfonamides;
widespread resistance by 1940s
Still sensitive to penicillin; used for Rx until
1980s
Tetracylcines, fluoroquinolones and
aminoglycosides all available in 1940s to
1960s with gradual resistance observed
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
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Rapidly increasing fluoroquinolone resistance
in 1990s
Cephalosporin resistance in early 2000s
True ceftriaxone resistance in Japan in 20111
◦ Isolate of GC with MIC to ceftriaxone of 2-4 µg/mL
from individual in Kyoto
◦ Clinical response to ceftriaxone?
1. Ohnishi et al. Antimicro. Agents. Chemo. 2011.
55: 3538-45.
1. From Unemo and Shafer, Ann. N.Y. Acad. Sci.
2011. 1230: e19-28.

From 2007 to 2010:
◦ Ciprofloxacin R has increased from 2.0% to 29.2%
◦ Ceftriaxone and Cefixime both remain 100% S
◦ Note that selection bias is possible

Communication by Manitoba Health re:
updated PHAC guidelines1 for GC treatment
in December 2011:
◦ Recommended increased dose of cephalosporins
for treatment of GC (cefixime 800 mg x 1,
ceftriaxone 250 mg IM x 1) due to increasing
treatment failures
◦ No longer recommends fluoroquinolones
◦ Culture in MSM and TOC for all pharyngeal
infections, persistent SSx, alternative Tx regimens
and known contact with R GC
1. http://www.phac-aspc.gc.ca/std-mts/sti-its/alert/2011/alertgono-eng.php.

Surveillance of GC resistance patterns
◦ Will help identify high-risk groups for treatment
failure
◦ Will aid in guideline preparation

Origins hotly disputed:
◦ Imported from the Americas to Europe and Asia?
◦ Established in Europe but proliferated due to
urbanization?

First clinical descriptions in 1547 in the
Brevary of Health:
◦ Known as Morbus Gallicus or French pockes

Organism described in 1905, named

First serological tests developed in 1906
Spirochaeta pallida
◦ Determined the prevalence of syphilis1 in large
European urban centres to be 8-14%

Infamous Tuskegee Study of Untreated
Syphilis in the Negro Male:
◦ Between 1932 to 1962, 431 men followed
untreated to describe natural history of infection
1. Mandell, Principles and Practice
of Infectious Diseases, 2011.
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Rate of 0.4-0.6/100,000 in 1994 to 2000
Increased to 4/100,000 in 2008
Outbreaks of syphilis from coast-to-coast
including in Winnipeg:
◦ Primarily observed in MSM and sexworker
populations

Congenital syphilis:
◦ No cases in 2003 and 2004
◦ 8 cases in 2005, 7 in 2006, 8 in 2007, 7 in 2008
1. PHAC Canadian STI Guidelines, 2010

Direct detection:
◦ Smear of mucosal or
skin ulcer for
darkfield microscopy
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PCR is also possible
Culture (lab
animals)
Rapid tests


Primarily by serology
Two main classes of serological tests:
◦ Non-specific (VDRL, RPR)
◦ Specific (TP-PA, MHA-TP, CLIA, EIA)
Non-specific tests:
 VDRL
 RPR
 Used to follow response to disease
 Numerous false positives
Infectious:
 Lyme disease
 Rickettsial disease
 Mycobacterial
infection
 Malaria
 Leptospirosis
 Endocarditis
 Vaccination
Non-infectious:
 Pregnancy
 Blood transfusions
 Connective tissue
diseases (CTDs)
 Acute rheumatic
fever
 Chronic liver
disease
 Age
Treponemal-specific tests:
 TP-PA
 MHA-TP
 FTA-ABS
 Chemimicroparticle luminescent assay (CMIA)
and enzyme immunoassay (EIA)
 Persist for life
 Not associated with false-positives


Initial use of non-treponemal test (VDRL, RPR)
as screen
If reactive, proceed to treponemal specific
test to confirm infection with Treponema
pallidum
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
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Initial screen with treponemal specific
serological method
Reactive screens followed by non-treponemal
tests
Increased sensitivity in certain populations:
◦ HIV-positive
◦ Immigrants/refugees

Possible sensitivity issues in early infection
1. Point Counter-point, J. Clin. Micro. Jan, 2012.
50(1): 2-6
Reverse screening:
 Equal to superior
sensitivity to RPR
 Better specificity
 Overall process more
sensitive
 Automation
 May miss early
infection
Traditional screening:
 US CDC still advises
traditional screening
 May see less initial
screen positives
 Potential higher cost:
◦ Patient follow-up
◦ Overtreatment

More useful in low
prevalence; low
volume labs
1. Point Counter-point, J. Clin. Micro. Jan, 2012.
50(1): 2-6
RPR tests performed at CPL from 200o to 2011
70,000
60,000
Number of tests performed
50,000
40,000
Total
30,000
20,000
10,000
2000
2001
2002
2003
2004
2005
2006
Year of test
2007
2008
2009
2010
2011
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
Currently using a traditional screening
algorithm
Limited use of reverse screening algorithm
for certain populations:
◦ HIV-positive
◦ Immigrant/refugee

Moving to exclusively reverse screening in
2012:
◦ Good results in Ontario, Alberta and Quebec
Adults and children estimated to be living with
HIV, 2008
Western & Eastern Europe
Central Europe & Central Asia
850 000
North America
1.4 million
[1.2 – 1.6 million]
Caribbean
240 000
[710 000 – 970 000]
Middle East & North
Africa
[220 000 – 260 000]
Latin America
2.0 million
[1.8 – 2.2 million]
1.5 million
[1.4 – 1.7 million] East Asia
310 000
[250 000 – 380 000]
Sub-Saharan Africa
22.4 million
[20.8 – 24.1 million]
850 000
[700 000 – 1.0 million]
South & South-East
Asia
3.8 million
[3.4Oceania
– 4.3 million]
59 000
[51 000 – 68 000]
Total: 33.4 million (31.1 – 35.8 million)
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64,800 HIV infections in Canada from 1985
to 2008
517 Canadian children; most by mother-tochild-transmission (MTCT)
Aboriginals account for 23% of new infections
Approximately 15% of HIV-exposed infants
Aboriginal
25% unaware of their HIV-positive status
From 1985 to January 20101:
1.
1682 Manitobans have been diagnosed with
HIV
2.
Women comprise 454 (27%) of those tests
3.
Majority of women between 15 and 39 years
of age (364 or 80.2%)
1. MHHL Stastical Update on HIV/AIDS, January 1, 1985 to
December 31, 2007 and
http://www.gov.mb.ca/health/publichealth/cdc/surveillance/ind
ex.html

1st generation EIAs:
◦ HIV antigen from infected T-lymphocytes
◦ False positive reactions due to HLA

2nd generation EIAs:
◦ Recombinant HIV antigen from viral or yeast vectors
◦ Prone to false-positive from reaction to vector
antigens

3rd generation EIAs:
◦ Synthesized HIV antigen, high purity with little cross
reactivity
◦ First to detect IgG, IgM and IgA

4th generation EIAs:
◦ Detection of both patient antibodies and p24
antigen
◦ Also detects IgG, IgM and IgA
◦ Available in Canada; introduced in US in October
2010
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1G
2G
3G
4G
EIA:
EIA:
EIA:
EIA:
+60 days
+40 days
+20-25 days
+15 days

Western Blot:
◦ Used if screen EIA tests
positive
◦ Detects patient
antibodies to HIV
proteins
◦ Various interpretive
criteria
◦ Most use combination
of:
 p24, gp41, gp120/160
◦ Considered
indeterminate if only 1
band positive
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1G EIA: +60 days
2G EIA: +40 days
Western blot: +30 days
3G EIA: +20-25 days
4G EIA: +15 days
HIV DNA assays:
 Detects proviral HIV
from PBMCs
 Excellent sensitivity
and specificity
 Not as widely
available as RNA
based tests
HIV RNA assays:
 Used in regular HIV
follow-up
 Either RT-PCR based
or branched-chain
DNA amplification
 May have limited
sensitivity (25-50%)
in first 72 hours of
life (Read, 2007)
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1G EIA: +60 days
2G EIA: +40 days
Western blot: +30 days
3G EIA: +20-25 days
4G EIA: +15 days
HIV DNA NAAT: +10-15 days
HIV RNA NAAT: +7-10 days
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Rapid serological tests available
All have comparable sensitivities and
specificities to ELISA/EIA
◦ Sensitivity 99.3-100%, Specificity 99.1-100%
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

Results available in ~15 to 30 minutes
Requires confirmatory testing if positive result
obtained
Based on:
◦
◦
◦
◦
Immunofiltration
Immune chromatography
Immunodot
Particle agglutination
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1G EIA: +60 days
2G EIA: +40 days
Western blot: +30 days
3G EIA: +20-25 days
POCT: +20-25 days
4G EIA: +15 days
HIV DNA NAAT: +10-15 days
HIV RNA NAAT: +7-10 days

CDC (2006):
 Opt-out screening
 All people 13 to 64 years
regardless of risk
 Repeated at least annually
if considered at risk
 Repeat screening if
presenting with STI
complaints
 Screening not required if
<1 HIV Dx per 1000 tested
 Adopted by the WHO in
2007

Canadian STI Guidelines
(PHAC 2008):
 Screening based on riskfactors
 Informed consent required
with pre and pos-test
counselling
Total HIV EIA/WB tests performed in MB from 2000 to
2011
70,000
Number of tests
60,000
50,000
40,000
30,000
Total Tested
20,000
10,000
2000
2001
2002
2003
2004
2005
2006
Year of test
2007
2008
2009
2010
2011
Positive and indeterminate HIV testing trends in MB 2000
to 2011
200
180
160
140
120
Indeterminate
100
80
Positive
60
Positivity per 10,000
40
20
2000
2001
2002
2003
2004
2005
2006
Year of test
2007
2008
2009
2010
2011
Positivity
Indeterminate Positive Total Tested per 10,000
Percent
Positive
Year
Negative
Percent I+P
2000
25,752
11
81
25,844
31.3
0.3
0.36
2001
27,037
15
88
27,140
32.4
0.3
0.38
2002
29,643
38
87
29,768
29.2
0.3
0.42
2003
32,495
20
129
32,644
39.5
0.4
0.46
2004
35,797
36
150
35,983
41.7
0.4
0.52
2005
38,706
36
141
38,883
36.3
0.4
0.46
2006
41,453
46
106
41,605
25.5
0.3
0.37
2007
45,537
25
104
45,666
22.8
0.2
0.28
2008
51,930
28
113
52,071
21.7
0.2
0.27
2009
55,463
27
176
55,227
31.9
0.3
0.36
2010
58,526
20
182
58,850
30.9
0.3
0.34
2011
63,349
21
125
63,554
19.7
0.2
0.23
Year
Syphilis Testing
GC Testing Total HIV Testing Total
Total
2000
30,102
25,844
34,012
2001
34,180
27,140
33,612
2002
41,796
29,768
34,385
2003
51,088
32,644
39,596
2004
59,613
35,983
43,055
2005
66,265
38,883
45,450
2006
74,991
41,605
48,104
2007
85,264
45,666
51,025
2008
99,048
52,071
54,770
2009
101,537
55,227
56,667
2010
99,507
58,850
54,327
2011
107,433
63,554
60,269

Since April 2008, HIV POCT has been
introduced or trialed at 3 sites in Manitoba:
◦ Nine Circles Community Health Centre (NCCHC)
◦ Women’s Hospital HSC
◦ Adult Emergency Department HSC

NCCHC
◦ Introduced in April 2008
◦ From April 2008 to December 2011, 2191 POCTs
performed.
◦ 22 (1.00%) individuals had reactive POCTs that were
confirmed HIV positive.
◦ An additional 2 individuals were indeterminate
following confirmatory serological testing

Women’s Hospital HSC
◦ Introduced in January 2009
◦ From January 2009 to December 2011, 90 POCT
have been administered on women receiving care at
the WH (11 tests in 2009, 21 in 2010 and 59 in
2011)
◦ 1 reactive in December 2011 which led to the
appropriate prevention of HIV mother-to-childtransmission (PMTCT) protocol being initiated

Adult ED at HSC
◦ Pilot study to determine feasibility of administering
POCT
◦ 501 POCT were performed from October 2010 to
October 2011
◦ 7 POCT were reactive (1.4%) and confirmed HIVpositive by standard serological methods at CPL
Advantages:
a)
Rapid assessment of pregnant women
considered at high-risk of HIV for initiation of
PMTCT
b) Immediate linking to HIV care of transient, highrisk individuals should their screening test
return reactive.
c)
Delivery of HIV screening in remote
communities, particularly in the developing
world.
d) Healthcare or non-healthcare exposure to
suspected HIV-positive individual.
Disadvantages:
a) Cost of POC testing is approximately
$15/test (Canadian) versus $1.78/test for
standard serological screening.
b) Proficiency and quality may not be possible
if few tests are performed per site or
multiple operators are performing POCT.
c) Psychosocial barriers to testing are equally
present with POCT as serological screening
in northern and rural communities.

Pilot of POCT in Bruntwood RHA:
◦ ED and labour floor



Discussion of POCT in NOR-MAN RHA
Evaluation of data to determine additional
sites based on high prevalence
Further refinement of delivery of POCT on a
provincial scale:
◦ To discuss with Ontario, Alberta, Saskatchewan and
BC
1.
2.
3.
4.
Provide a general overview of diagnostic
microbiology testing
Review the evolution of resistance in
Neisseria gonorrhea and its impact on
testing
Compare the traditional and reverse
screening algorithms for syphilis
Discuss HIV testing, in particular the role
and challenges of HIV POCT