Molecular diagnosis and techniques for bacterial STIs in a West

ÖREBRO LÄNS LANDSTING
N. gonorrhoeae
T. pallidum
C. trachomatis
Molecular diagnosis and techniques for bacterial
STIs in a West-European Reference Laboratory
Magnus Unemo, Assoc. Professor, WHO Collaborator
National Reference Laboratory for Pathogenic Neisseria
Örebro University Hospital, Sweden
ÖREBRO LÄNS LANDSTING
Nucleic acid amplification tests (NAATs)
• Latest one-two decades, NAATs revolutionized diagnostics
of infectious diseases, including STIs
• Dramatic improvement in sensitivity, and mostly specificity
• Allow automation, non-invasively collected specimens
and screening of asymptomatic individuals ⇒ reliable
population based studies, determine incidence/prevalence,
and eliminate the reservoir of infection
• All NAATs are not equal ⇒ crucial with strict selection,
validation (sampling  report), and quality assurance (QA)
and quality control (QC; internal and external)
• NAATs do not solve all the problems, and phenotypic
methods remain essential for several issues!
ÖREBRO LÄNS LANDSTING
Diagnosis of gonorrhoea
(Unemo. Swedish Ref. Meth., ~internationally)
• Clinical: only suggestive for bacterial STIs!
Laboratory diagnostics
• Microscopy of Gram-stained smear: mainly presumptive
(if not urethral specimen from symptomatic male)!
• Culture (optimized and quality assured): Gold standard for
definitive diagnosis (”100%” specificity and AMR testing!)!
• DNA/RNA-based assays: Confirmation required for
definitive diagnosis of all samples
(specificity problem and no AMR data)!
Effective NAAT (NEW GENERATION!), confirmation,
where suboptimal culture, screening, asympt...!!!
ÖREBRO LÄNS LANDSTING
Internationally validated commercial
DNA/RNA-based diagnostic assays
Genetic target
Method
Manufacturer
16S rRNA
Probe hybridisation
Gen-Probe
Chromosomal and plasmid sequences
Probe hybridisation
Digene Corporation
cytosine DNA methyltransferase gene
(single-copy)
PCR
Roche Diagnostics
PCR (m2000)
LCR (LCx)
Abbott Laboratories
Abbott Laboratories
NASBA
Organon
teknika/BioMerieux)))
TMA Target capture
Gen-Probe
SDA
Becton Dickinson
opa genes (multi-copy)
opa genes
(((16S rRNA (multi-copy)
16S rRNA (multi-copy, different segment
for confirmation)
pivNG gene (multi-copy)
N. subflava, N. cinerea, N. flavescens, N. lactamica, N. sicca, etc genetically very
similar ⇒ false positives ⇒ confirmation using other target(s) mainly needed!
In house targets: PCR: cppB gene, porA pseudogene, opa genes, LCR: pilin gene
ÖREBRO LÄNS LANDSTING
Nucleic acid amplification tests (NAATs)
Advantages

mostly superior sensitivity compared to culture,
especially for pharyngeal and rectal specimens

rapid and possibilities to high automation

non-invasive specimens, e.g. urine (⇒ screening)



opportunities for self- and home-sampling, and even
cost-efficient pooling if the prevalence is not too high
(e.g. screening and epidemiological surveillance) (Lindan,
et al. 2005. JCM:1674-7)
no requirements of viable bacteria (only DNA/RNA)
opportunities for simultaneous detection of several
agents, e.g. Chlamydia trachomatis and GC
ÖREBRO LÄNS LANDSTING
Positive Predictive Value (%)
The specificity problem ⇒ several NAATs have exceedingly low
PPVs in low prevalent populations (relate to prevalence)!
100
Test sensitivity 90%
90
1% (still high) infection prevalence
80
70
Sensitivity 88.1% and specificity 99.2%
Specificity = 99.0
(mean results for men in Cook, et al. 2005. AIM:914-25)
⇓
Specificity = 98.0
Positive predictive value (PPV) of only ≈53%
Specificity = 97.0
Confirmation needed!
60
50
40
30
20
10
0
0
2
4
6
8
10 12 14 16 18 20
Prevalence (%)
ÖREBRO LÄNS LANDSTING
Disadvantages




Specificity problems with many methods (e.g. false positive
samples especially in pharyngeal and rectal specimens,
confirmative assay(s) required!)
Sensitivity problems with some mainly in house methods (due
to the choice of target and/or presence of inhibitors])
Expensive equipment and reagents
Risk of amplicon (DNA/RNA) contamination, present all over
the sampling clinic and laboratory (Meader, et al. 2008. STI:107-10)?,
additional well-designed studies are needed!

Not effective?! and licensed for extragenital specimens!

Comprehensive and reliable AMR testing not possible!
NEW generation NAATs (APTIMA Combo 2, Abbott
m2000, Cobas 4800, BD Viper, [Siemens]): higher
specificity, currently introduced by the companies!
ÖREBRO LÄNS LANDSTING
Diagnosis of genital chlamydia
(C. trachomatis)
- Clinical:
only suggestive (even if symptomatic)!
Laboratory diagnostics
• NAATs
•
•
•
•
•
•
•
Culture in viable cell lines (McCoy, Hela 229, BGMK –
fluorescence labelled antibodies [MOMP/LPS])
100% specificity?
Direct fluorescent antibody (DFA) tests [MOMP/LPS]
Enhanced enzyme immunoassay (EIA) tests [MOMP/LPS]
EIA [MOMP/LPS]
Rapid tests (”Point-of-care”) [MOMP/LPS]
Serology
Microscopy
Decreased
sensitivity
Unemo, Papp. Atlas of STDs and AIDS. 2010
ÖREBRO LÄNS LANDSTING
Swedish new variant of CT (nvCT)
- Deleted plasmid target in the Roche and Abbott PCRs
(Ripa. Euro Surveill. 2006) ⇒ thousands of false negatives
- nvCT plasmid characterized
(Seth-Smith, et al. BMC Genomics. 2009)
- Dual-target assays from Abbott and Roche in 2008
- nvCT completely characterized
(genome sequenced, complete phenotypic
analysis ⇒ NO enhanced biological fitness
and only diagnostic selective advantage!)
(Unemo, et al. Microbiology. 2010)
ÖREBRO LÄNS LANDSTING
Internationally validated commerical
DNA/RNA-based diagnostic NAATs
Genetic target
Test/system
Manufacturer
Cryptic plasmid
Cobas Amplicor
Roche
Diagnostics
Cryptic plasmid (+ompA)
Cobas TaqMan48,
4800
Roche
Diagnostics
Cryptic plasmid (+plasmid)
m2000
Abbott
Laboratories
Cryptic plasmid
BD ProbeTec/Viper
Becton Dickinson
23S rRNA (16S confirm.)
Aptima
Combo 2
Gen-Probe
ompA (omp1) gene
artus
Qiagen
ompA gene +
artus Plus
Qiagen
cryptic plasmid
VERSANT
Siemens CT/GC DNA 1.0, Siemens
Unemo, Papp. Atlas of STDs and AIDS. 2010
ÖREBRO LÄNS LANDSTING
Routine CT diagnostics in our Reference Lab.

Culture
(1/10 of samples,
only laboratory in Sweden
still using also culture)

AMR testing, nvCT
and other mutants,
genome studies,
phenotypic assays,
other research
PCR
(9/10 of samples)
TaqMan48, Cobas Amplicor,
LightCycler480 (Unemo. Euro
Surveill. 2007), Roche Diagnostics
ÖREBRO LÄNS LANDSTING
C trachomatis multicenter 3832 patienter
100
90
80
70
60
50
40
30
20
10
0
Sensitivity clinical samples (3 groups):
GenProbe Aptima Combo (AC2)
>
AC2
Abbott m2000 >=
Roche (Cobas, Taqman48)
>
BD ProbeTec
sens
Abbott
m2000
BD
ProbeTec
spec
Möller, et al. JCM. 2010; Masek, et al. JCM. 2009; Walsh, et al. DMID. 2009…..
ÖREBRO LÄNS LANDSTING
Diagnosis of syphilis
Natural course of untreated syphilis
6 weeks
to
6 months
Approx.
18 months
Primary
(Chancre)
Secondary
(Rash)
Many years
to a lifetime
Latent Syphilis
(No signs of disease)
Tertiary
Benign gummatous
Cardio-vascular syphilis
Neurosyphilis
Incubation period
9 – 90 days
1-2 years
Many years
to a lifetime
Early Syphilis
Late Syphilis
ÖREBRO LÄNS LANDSTING
DFA-TP, direct fluorescent antibody-T. pallidum; RPR, rapid plasma reagin; MPR, microprecipitation reaction;
VDRL, Venereal disease research laboratory; TPHA/TPPA, TP haemagglutation/passive particle
agglutination; FTA-ABS, fluorescent treponemal antibody-absorption
Sokolovskiy, et al. JEADV. 2009
ÖREBRO LÄNS LANDSTING
Syphilis diagnostics in our Ref. Lab., as in many
other low prevalent ”western” countries
• Architect CMIA* (automated ~treponemal ELISA)
– screening in low prevalent population!
• VDRL/RPR
• TPPA
+
+
+
+/- -
• PCR (bmp gene; ulcer!)
-
Verification and
follow-up
- PCR (bmp; ulcer!)
• (Rapid tests)
*Chemiluminiscent microparticle immunoassay
- Captiva IgM
- Wasserman
Foto: Jens Blom, Statens Seruminstitut
ÖREBRO LÄNS LANDSTING
Diagnosis of M. genitalium infection
• Culture:
lacks sensitivity and very time consuming
• Serology: lacks specificity and sensitivity
• PCR and other NAATs are the only effective and practical
methods
- Target genes:
- MgPa adhesin gene (real-time PCR in routine)
- 16S rRNA gene (if required, confirm., discrepancy)
- No commercially available assays ⇒ strict validation
- Technically complicated (14% of urines contain <400 geq/ml)
- Evidence-based choice of NAAT and strict validation, QA,
and QC crucial
(Shipitsyna, et al. ADV. 2010)
ÖREBRO LÄNS LANDSTING
Molecular additional methods for confirmation,
characterization, and research in our Ref. Lab.
• N. gonorrhoeae:
- two in house real-time PCRs for diagnostics
- five different epidemiological typing methods
- methods for studying 23 different resistance determinants
• C. trachomatis:
- one commercial PCR, and three in house PCRs for
diagnostics
- three different epidemiological typing methods
• T. pallidum:
- one in house PCR for diagnostics
- one epidemiological typing methods
- one method for detection of azithromycin resistance
• M. genitalium:
- two in house PCRs for diagnostics
- one epidemiological typing method
ÖREBRO LÄNS LANDSTING
Conclusions
• NAATs have dramatically improved the sensitivity, mostly the
specificity, speed, and accessibility of asymptomatic patients in
the STI diagnostics
• All NAATs are not equal ⇒ crucial with strict selection,
validation to international approved systems, and QA and QC
(Detailed Guidelines!)
• NAATs need to be combined with optimized, validated, and QA
and QC phenotypic methods for several STIs, e.g. gonorrhoea
(culture and AMR testing) and syphilis (serology)
• Future?:
- Broad multiplex NAATs (multiple, multicopy, housekeeping
targets and/or multiple agents)
- Point-of-care NAATs; diagnosis, screening of AMR mutation, and
typing
- Microarray technology, genome sequencing, backscatter
interferometri, MaldiTof MS, and nanotechnology?
ÖREBRO LÄNS LANDSTING
ÖREBRO LÄNS LANDSTING
CT elementary bodies in clinical specimens
(vaginal swabs: for screening but also for diagnostics)
Michel, et al, JCM, 2007.
More ref`s: Backen, 2005; Skidmore, 2006; Fang, 2008; Falk, 2010