GRF 2nd One Health Summit 2013: Presentation by Rudy Hartskeerl, WHO/FAO/OIE and National Leptospirosis Reference Centre

1. Diagnosis of leptospirosis
Rudy A. Hartskeerl
WHO/FAO/OIE and National Leptospirosis Reference
Centre

2. Clinical manifestations
•
Large variety of signs and symptoms mimicking several
other diseases (dengue, hanta virus, yellow fever, hepatitis,
salmonellosis, brucellosis, malaria, pneumonia)
•
Asymptomatic, mild (flu-like) to severe course
• Weil’s syndrome (kidney failure, liver impairment (jaundice),
hemorrhage: CFR 10-20%
• Severe Pulmonary Hemorrhage Syndrome (SPHS): globally
emerging form with CFR 50-80%
•
Diagnosis on clinical grounds is challenging
Amsterdam, The Netherlands
www.kit.nl

3. Conventional tests
Conventional tests: DFM, MAT, ELISA, IFAT, IHA, MSAT
etc.
Many drawbacks: slow, unreliable, low detection
threshold, difficult to standardize, require well-trained
personnel and/or expensive media/equipment
Isolation confirms leptospirosis but is too slow for
individual diagnosis and has mainly epidemiological value
Amsterdam, The Netherlands
www.kit.nl

4. Laboratory diagnosis
Serological tests
MAT
Gold or reference standard
Requires panels of ‘locally’ representative serovars
May provide indication for infecting serogroup
ELISA
IgM antibodies against leptospires
Genus specific
In-house ELISA: dilution titers
Both tests are restricted to well equipped expert labs
Amsterdam, The Netherlands
www.kit.nl

5. Sensitivity of MAT and IgM ELISA according to
stage of disease
Main findings
• Serology does not
contribute to confirmation
at early acute phase
• ‘Gold standard’ MAT
has sensitivity of 80%
• Use of paired samples
greatly improves
case detection
Amsterdam, The Netherlands
www.kit.nl

6. Rapid Diagnostic Tests for use at ‘peripheral’ level
Test/kit
Manufacturer
Technology
comments
IHA
Mayo Medical Lab
(USA)
Indirect
hemagglutination
Leptospira-MC test
Japan Lyophilization
Lab
Microcapsule
agglutination test
LeptoTek LFA
Organon Teknika
(Netherlands)
Lateral flow assay
Expected soon by
IMACCESS
Test-it
Life Assays (South
Africa)
Lateral flow assay
Human and
veterinary
Leptocheck-WB
Zephyr (India)
Lateral flow assay
SD Leptospira LF
Standard Diagnostics
(Korea)
Lateral flow assay
IgM LFA
Omega Diagnostics
(UK)
Lateral flow assay
LeptoTek DriDot
BioMerieux (France)
Latex cardagglutination test
Leptorapide
Linnodee (Northern
Ireland)
Latex cardagglutination test
Not available
GLEAN paper: Picardeau et al., Rapid tests for diagnosis of leptospirosis: current tools and emerging technologies.
Diag Microbiol Infect Dis (2013); Table 1
Amsterdam, The Netherlands
www.kit.nl

7. Leptocheck WB (Zephyr); sensitivity 2005-2011
100
90
80
70
60
%
50
40
30
20
10
0
"2005"
"2006"
"2009"
"2010"
"2011"

8. Drawbacks rapid tests
•
Confirmation by conventional tests is required
• Relatively low diagnotic accuracy
• Varying diagnostic accuracy => repeated local
evaluations
• Need for improved QA at production and/or test
performer (easy does not exclude experience)
•
Based on serology: too late for early diagnosis and
start of effective antibiotic treatment
•
Rapid diagnosis is different from early diagnosis!
Amsterdam, The Netherlands
www.kit.nl

9. Importance of high quality laboratories
Adequate laboratory capacity is
indispensible for:
•
Early detection of diseases
•
Disease surveillance
•
Evidence-based diagnostic
decision making
Amsterdam, The Netherlands
www.kit.nl

10. Quality Management System
Implementation of a quality management
system (QMS) is a key intervention to break
the negative spiral
•
•
www.kit.nl
The internationally accepted
quality standard for medical
laboratories is ISO 15189
•
Amsterdam, The Netherlands
The requirements for a wellfunctioning QMS are
formulated in quality
standards
A laboratory is accredited
when it complies with the
complete standard

11. Observations
The problem is that quality standards
indicate what shall be implemented,
not how it must be implemented.
Observations:
• Interest of laboratories to become
accredited is increasing
• It is difficult to translate the international
quality standard into practice
Amsterdam, The Netherlands
www.kit.nl

12. Electronic guidance tools
•
•
•
•
GLI tool: Global Laboratory Initiative
Stepwise Process towards TB Laboratory
Accreditation
Tool translate the ISO 15189 quality
standard into:
• Sequence of practical steps for
implementation
• Links to support materials
(trainings, templates, …) where
appropriate
Available online-www.GLIquality.org
Free of charge
Amsterdam, The Netherlands
www.kit.nl

13. Electronic guidance tools
WHO LQSI tool: World Health Organization Laboratory Quality
Stepwise Implementation tool – last stages of review.
Amsterdam, The Netherlands
www.kit.nl

14. In the context of OneHealth
LQSI tool and GLI tool focus on medical
laboratories.
To prevent problems related with global
urbanization other laboratories need to be
of adequate capacity as well:
•
•
•
•
•
Food security laboratories
Industrial laboratories
(pharmaceutics/food production/etc.)
Veterinary laboratories
Forensic laboratories
Etc.
Amsterdam, The Netherlands
www.kit.nl
ISO 15189
Similar tools needed for
ISO 17025: Test and
Calibration laboratories

15. Early diagnosis
Application of current rapid diagnostic tests (RDT) does not
provide early diagnosis!
Amsterdam, The Netherlands
www.kit.nl

16. Early diagnosis
•Early diagnostic gap
with serology
•Leptospires circulate in the blood notably first 4-7 days
after onset of disease: Several NA amplification
techniques on blood samples like PCR can fill in this gap
Amsterdam, The Netherlands
www.kit.nl

17. Hand held PCR equipment
•
•
•
•
•
•
•
Battery-powered stand-alone device:
More than 4 hours of operation on a single charge
High amplification speed:
Extreme high efficiency:
Near single-copy amplification sensitivity
Dynamic range sufficient for genetic identification:
Unmatched well-to-well uniformity:
Simple and adaptable:
Easy to program and compatible with standard tools
Affordable costs
Amsterdam, The Netherlands
www.kit.nl
T-COR 4 qPCR
Palm PCR F1-12

18. Future (tests)
•
Early, easy and rapid – antigen detection based?
•
Same test for humans and animals
•
Discriminative (multiplex testing/differential diagnosis)
•
Traceable (online data transfer to e.g. MoH and MoA) - early
outbreak warning (RDT with QR/barcodes or chip; data and
location recording by smart phone?)
Amsterdam, The Netherlands
www.kit.nl

19. Future tests: Antigen detection
Antigen test sens 102-103/ml – improve by use of nanobiosensors?
Isothermal Recombinase Polymerase Amplification (RPA):
multiplex, easy design (37-42oC, runtime 30 min, PCR-like
primers/probes)
10
-2
10
-3
10
-4
Piepenburg et al. (2006). DNA detection using recombination proteins. PLoS Biol 4:e204
Amsterdam, The Netherlands
www.kit.nl
10
10
10
-5
-6
-7
10
-8
H2
O
Pos.
C.

20. Quantification market for RDTs in Indonesia
Incidence
50 per
100,000
10-20% of
patients with
compatible
symptoms have
lepto
120.000 people
acquire
leptospirosis/ yr
7 x 120.000 =
840.000
patients/yr
require
leptotest
2 tests per
patient (paired
samples) =
1.680.000
tests required
population
240.000.000
Market might be increased in case of multiplex testing with
more common infectious diseases in differential diagnosis!

21. Summary
•
•
•
•
•
•
•
Leptospirosis is difficult to diagnose in the clinic: laboratory
support is indispensable
Conventional diagnostic tests are inadequate
RDT are helpful but ‘are late’, lack diagnostic accuracy and
consistency
Early and rapid diagnosis is possible with molecular antigenbased tests: applicable on both humans and animals
Test results should be digitally transmittable and associated
with data, important for surveillance and early outbreak
warning
Implementation of QA at several stages is badly needed
There is a potential market for leptospirosis RDT notably when
associated with multiplex testing for differential diagnosis
Amsterdam, The Netherlands
www.kit.nl

22. Thanks