Jared Bullard MD FRCPCPaediatric Infectious Diseases & Medical Microbiology

Associate Medical DirectorCadham Provincial Laboratory

WRHA STBBI ConferenceMarch 14, 2012

No conflicts of interest to declare

1. Provide a general overview of diagnostic microbiology testing

2. Review the evolution of resistance in Neisseria gonorrhea and its impact on testing

3. Compare the traditional and reverse screening algorithms for syphilis

4. Discuss HIV testing, in particular the role and challenges of HIV POCT

1. Chase children2. Bath and feed children3. Read MSc papers about lymphoma4. Associate Medical Director at Cadham

Provincial Laboratory5. Pediatric Infectious Diseases consultant6. Eat7. Chase children8. Sleep?

Host-based testing Pathogen-based testing

Antibody generated to pathogen Two main classes used in diagnosis:

◦ IgM (typically seen between 7-10 days post-exposure)

◦ 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:◦ 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

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 Thayer-Martin 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 15-

24 years Contrast to number of isolates by culture

analyzed at CPL:◦ 213 isolates referred between 2007-2010◦ 385,356 urine NAATs over same time

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, e-pub December 2011.

Preferred regimen: Cefixime 400 mg PO x 1 doseAlternative 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

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

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/alert-gono-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 Spirochaeta pallida

First serological tests developed in 1906◦ 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.

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

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

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

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

Total: 33.4 million (31.1 – 35.8 million)

Western & Central Europe850 000850 000

[710 000 – 970 000][710 000 – 970 000]Middle East & North

Africa310 000310 000

[250 000 – 380 000][250 000 – 380 000]Sub-Saharan

Africa22.4 million22.4 million

[20.8 – 24.1 million][20.8 – 24.1 million]

Eastern Europe

& Central Asia1.5 million 1.5 million [1.4 – 1.7 million][1.4 – 1.7 million]

South & South-East Asia

3.8 million3.8 million[3.4 – 4.3 million][3.4 – 4.3 million]Oceania

59 00059 000[51 000 – 68 000][51 000 – 68 000]

North America1.4 million

[1.2 – 1.6 million]

Latin America2.0 million2.0 million

[1.8 – 2.2 million][1.8 – 2.2 million]

East Asia850 000850 000

[700 000 – 1.0 million][700 000 – 1.0 million]Caribbean

240 000[220 000 – 260 000]

Adults and children estimated to be living with HIV, 2008

64,800 HIV infections in Canada from 1985 to 2008

517 Canadian children; most by mother-to-child-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 HIV2. Women comprise 454 (27%) of those tests3. 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/index.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

1G EIA: +60 days 2G EIA: +40 days 3G EIA: +20-25 days 4G EIA: +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

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)

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

Rapid serological tests available All have comparable sensitivities and

specificities to ELISA/EIA◦ Sensitivity 99.3-100%, Specificity 99.1-100%

Results available in ~15 to 30 minutes Requires confirmatory testing if positive

result obtained Based on:

◦ Immunofiltration◦ Immune chromatography◦ Immunodot◦ Particle agglutination

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 screeningAll people 13 to 64 years

regardless of riskRepeated at least annually

if considered at riskRepeat 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 risk-

factorsInformed consent required

with pre and pos-test counselling

Year Negative Indeterminate Positive Total TestedPositivity

per 10,000Percent Positive 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 GC Testing

TotalHIV Testing

TotalSyphilis Testing

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-child-transmission (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 HIV-

positive 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, high-risk 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. Provide a general overview of diagnostic microbiology testing

2. Review the evolution of resistance in Neisseria gonorrhea and its impact on testing

3. Compare the traditional and reverse screening algorithms for syphilis

4. Discuss HIV testing, in particular the role and challenges of HIV POCT