Activities in 2009
Dr R.A. Hartskeerl
OIE Reference laboratory for Leptospirosis, KIT Biomedical Research
Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands
Tel.: (+31-20) 566.5438, Fax: (+31-20) 697.1841
E-mail address: email@example.com
Summary of general activities related to the disease
1. Test(s) in use/or available for the specified disease at your laboratory
Test For Specificity research in
(diagnosis in humans)
ELISA Antibody Genus 601 0
Microscopic Antibody Serogroup 601 69
Culture Antigen Serovar 180 0
Real time PCR DNA Pathogenic Leptospira 112 2
Animals. For the sero-diagnosis in animals, the centre received sera from 45 mice, 19 cows, 2 dogs, 2 sea lions and
1 lion. Investigations of the sera from the mice concerned requests for a quality check. All sera were negative.
PCR (negative) was performed on tissue samples from the lion and a brown rat. Except for the sea lions (USA), all
animals were from the Netherlands.
Humans. Human diagnosis forms the basis for the national surveillance task. In 2009, a total of 656 (compared to
787 in 2008) human specimens were tested. In 24 cases (20 males, 4 females) the diagnosis leptospirosis was
established. In 3 cases this was based on a positive real time PCR. To our knowledge 1 patient died (only positive
in PCR). Five infections acquired in The Netherlands were due to occupational activities, with 2 potentially
associated with contact to farm animals. For 1 additional infection acquired in The Netherlands the probable cause
is unknown. In addition, 17 sera from the USA and 5 from Finland have been tested by MAT and IgM ELISA and
53 serum samples from the outbreak in Sri Lanka have been tested by MAT. PCR on a set of 28 samples from
patients without microbiological diagnosis obtained from a local academic hospital revealed 3 (10.7%) positive
samples stressing that under-diagnosis also poses a major problem in The Netherlands
2. Production and distribution of diagnostic reagents
During the last decade, the reference centre has equipped more than 100 laboratories all over the world with
various reference materials, including Leptospira reference strains, (panels of) rabbit anti-Leptospira reference
sera and mAbs that enable these laboratories to quickly and easily identify most locally occurring strains. In 2009,
mAbs, Leptospira strains, culture media, ELISA reagents, rabbit anti-Leptospira sera, DNA, primers and probes
were sent to several institutions in Australia, Belgium, Brazil, Canada, China (Hong Kong), Costa Rica, Czech
Republic, Croatia, Cuba, Denmark, Germany, Indonesia, Ireland, Italy, Mexico, Nigeria, Romania, Slovenia,
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009 1
South Korea, Spain, Sri Lanka, Switzerland, Thailand, The Netherlands, United Kingdom and United States of
America. These are all OIE member countries.
Activities specifically related to the mandate
of OIE Reference Laboratories
3. International harmonisation and standardisation of methods for diagnostic testing or the
production and testing of vaccines
The centre has a CCKL accreditation (ISO 15189-based). This ISO 15189-based accreditation is most suitable for
the centre that also functions as National and WHO/FAO reference centre and forms the diagnostic centre for
human leptospirosis in The Netherlands. In a joint effort with the Leptospirosis Reference Unit, Department of
Microbiology and Immunology, County Hospital, Hereford and the National Serology Reference Laboratory
(NRL), Australia, Melbourne, the reference centre in Amsterdam performed the eight annual round of an
international proficiency testing of the MAT. Late 2009, 93 panels were sent to participants, mostly veterinary
institutions and (national) reference centres.
4. Preparation and supply of international reference standards for diagnostic tests or vaccines
The reference laboratory provides reference rabbit sera and mAbs for typing and checking of (diagnostic) strains
and provides backstopping to laboratories by performing quality control and typing of problem strains.
5. Research and development of new procedures for diagnosis and control
Research focuses on new simple and rapid serological methods and molecular tests for early diagnosis. Since
2004, a lateral flow test from Zephyr Biomedicals, India was included in a prospective evaluation. A preliminary
report over the period 2004-2007 was drafted to aid decisions making during the leptospirosis outbreak in the
Philippines. Further joint research within the framework of formal (externally financed) and informal
collaborations was aimed at the validation (following the OIE scheme) of a real time PCR for the detection of
Leptospira in human and animal specimens and of molecular typing tools, the unravelling of the pathogenesis of
leptospirosis and the surveillance and epidemiology of (animal and human) leptospirosis in several regions in the
6. Collection, analysis and dissemination of epizootiological data relevant to international disease
In 2009, the reference centre received or obtained 21 Leptospira isolates for typing as follows: 3 human isolates
from Sri Lanka, 2 rodent isolates from the Azores islands, 9 rat isolates from Madagascar, 6 isolates from various
origins from USA and 1 human isolate from The Netherlands. Two reference strains were received from Slovenia
for an identity check.
7. Provision of consultant expertise to OIE or to OIE Members
The centre participated in requests and questionnaires from OIE and other animal health related investigations.
In 2009, the centre (i) provided consultancy to WHO concerning the set-up of guidelines for diagnosis, treatment
and surveillance in South East Asia and the evaluation of a leptospirosis epidemic in the Philippines and (ii)
participates in the WHO Leptospirosis Burden Epidemiology Reference Group (LERG).
8. Provision of scientific and technical training to personnel from other OIE Members
In 2009, the centre did not contribute to an international leptospirosis course. The centre guided several MSc and
PhD students and trainees from Turkey, Malaysia, Slovenia, Romania and The Netherlands.
2 Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
9. Provision of diagnostic testing facilities to other OIE Members
See point 2 and 4.
Agent identification is carried out using rabbit sera against reference Leptospira strains and panels with
monoclonal antibodies (mAbs) that agglutinate serovars in a characteristic way. The Cross Agglutinin Absorption
Test (CAAT) is the gold standard for serovar determination. DNA determination is done by (species) specific
PCRs and gene-sequencing. Total number of isolates identified in 2009: 7 isolates, i.e. 3 isolates from Sri Lanka,
2 isolates from humans in The Netherlands and 2 isolates from rodents in Portugal.
Additionally, checks were performed on 2 reference strains from Slovenia, 3 strains from Czech Republic and 6
animal vaccine strains from The Netherlands.
10. Organisation of international scientific meetings on behalf of OIE or other international bodies
Co-organization of the international scientific Meeting of the International Leptospirosis Society, "LeptoCON"
2009, Cochin, India, 21-24 September 2009.
11. Participation in international scientific collaborative studies
The centre performed in 2009 the following project: ECDC public tender OJ/2009/05/19-PROC/2009/018
“Scientific input into the selection and processing of datasets for the European Environment and Epidemiology
(E3) network” started in October 2009. The OIE Reference Laboratory participates in the consortium for the
inclusion of leptospirosis as a typical water-borne disease.
12. Publication and dissemination of information relevant to the work of OIE (including list of
scientific publications, internet publishing activities, presentations at international conferences)
Presentations at international conferences and meetings
Presentation. Hartskeerl RA, Invited speaker. Leptospirosis: a prototype Neglected Infectious Disease. 70th
Anniversary Congress of Instituto Pedro Kouri, 2-6 June 2009, Havana, Cuba.
Presentation. Hartskeerl RA. Invited speaker. Burden of leptospirosis. WHO-SEA meeting 16-18 September 2009,
Presentation. Goris MGA, Wagenaar JFP, Hartskeerl RA, Gorp ECM van, Poll T van der, Veer C van 't. Innate
immune response to pathogenic and non-pathogenic Leptospira is dependent of both TLR2 and TLR4 signaling in
human whole blood. 6th International Leptospirosis Society Meeting "LeptoCON" 2009, Cochin, India, 21-24
Presentation. Hartskeerl RA, Invited speaker. Leptospirosis: Current status and future trends in human
leptospirosis. Aravind Eye Hospital, Madurai, India, 26-27 September 2009.
Presentation. Hartskeerl RA. Invited speaker. Human Leptospirosis at a global perspective.. ISC conference
Emerging Zoonotic Diseases, 13-18 October 2009, Cairo, Egypt.
Poster. Ahmed A, Engelberts MFM, Ahmed N, Hartskeerl RA. Development and validation of a real-time PCR for
the early diagnosis of leptospirosis. 6th International Leptospirosis Society Meeting "LeptoCON" 2009, Cochin,
India, 21-24 September 2009.
Poster. Barragan VA, Mejia ME, Zapata S, Travez A, Haake DA, Hartskeerl RA, Trueba GA. Enviromental
Bacteria Foster the growth of Saprophytic but not Pathogenic Leptospira. 6th International Leptospirosis Society
Meeting "LeptoCON" 2009, Cochin, India, 21-24 September 2009.
Poster. Carmona-Gasca CA, Hartskeerl R, Ahmed N, Lemuel A, Lara L, De la Pena-Moctezuma A. Evaluation of
gspD in MLST scheme for the identification of Leptospira isolates. 6th International Leptospirosis Society
Meeting "LeptoCON" 2009, Cochin, India, 21-24 September 2009.
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009 3
Poster. Chappel RJ, Goris MGA, Palmer MF, Hartskeerl RA. Lessons Learned from the Results of Proficiency
Testing for the Leptospirosis Microscopic Agglutination Test. 6th International Leptospirosis Society Meeting
"LeptoCON" 2009, Cochin, India, 21-24 September 2009.
Poster. Galloway RL, Buchholz AE, Whelen AC, Zaanen van W, Hartskeerl RA. Characterization of Leptospira
isolates from patients in Hawaii using molecular and serological techniques. 6th International Leptospirosis
Society Meeting "LeptoCON" 2009, Cochin, India, 21-24 September 2009.
Poster. Munoz-Zanzi, C, Neils L, Hrnickel L, Belongia E, Hartskeerl R, Lesina B. Estimation of sensitivity and
specificity of screening tests for leptospirosis in traditionally low risk populations. 6th International Leptospirosis
Society Meeting "LeptoCON" 2009, Cochin, India, 21-24 September 2009.
Poster. Rebeiro G, Gouveia EL,Almeida SF, Flannery B, Ramos JG, Pitangueira Filho JC, De Carvalho AL,
Villasboas Bisneto JC, Aires T, Queiroz A, Fraga DB, Santos AC, Salgado K, Reis RB, Hartskeerl R, Reis MG,
Ko A. Cyclic rainfall - Associated epidemics of severe leptospirosis in the urban setting. 6th International
Leptospirosis Society Meeting "LeptoCON" 2009, Cochin, India, 21-24 September 2009.
Poster. Zeng L, Guo Z, Liu Y, Zeng Y, Ahmed N, Hartskeerl RA. Surveillance of leptospirosis at SiChuan
Province in 2008. 6th International Leptospirosis Society Meeting "LeptoCON" 2009, Cochin, India, 21-24
Poster. Hopman J, Stelma FF, Ahmed A, Ven van der AJ, Brouwer AE, Keuter M, Hartskeerl RA, Becker PJ,
Galama JM, Sturm PD, Sauerwein RW. Microbiological analysis of travelers with febrile illness returning to the
Netherlands. 6th European Congress on Tropical Medicine and International Health, Verona, Italy, September 6-
Scientific publications in peer-reviewed journals
Houwers DJ, Wagenaar JA, Hartskeerl RA, Hautvast JLA, Stinis HPJ, Ruijs WLM, Lenaers SJMM. Leptospirose
(ziekte van Weil) bij de hond: een risico voor de mens? Tijdschrift voor Diergeneeskunde, 2009, 134, 392-394.
Wagenaar JFP, Goris MGA, Sakundarno A, Gasem H, Isbandrio B, Moalli F, Mantovani A, Boer KR, Hartskeerl
R, Garlanda C, Gorp ECM van. Long pentraxin PTX3 is associated with mortality and disease severity in severe
Leptospirosis. Journal of Infection Society, 2009, 58, 425-432.
Ahmed A, Engelberts MFM, Boer RK, Ahmed N, Hartskeerl RA. Development and Validation of a Real-Time
PCR for Detection of Pathogenic Leptospira Species in Clinical Materials. PLoS ONE, 2009, 9, 1-8
Wagenaar JF, Gasem MH, Goris MG, Leeflang M, Hartskeerl RA, van der Poll T, van 't Veer C, van Gorp EC.
Soluble ST2 Levels Are Associated with Bleeding in Patients with Severe Leptospirosis. PLoS Neglected Tropical
Diseases, 2009, 3, e453.
Gasem MH, Wagenaar JF, Goris MG, Adi MS, Isbandrio BB, Hartskeerl RA, Rolain JM, Raoult D, van Gorp EC.
Murine typhus and leptospirosis as causes of acute undifferentiated fever, Indonesia. Emerging Infectious
Diseases, 2009, 15,975-7.
Wagenaar JF, Goris MG, Partiningrum DL, Isbandrio B, Hartskeerl RA, Brandjes DP, Meijers JC, Gasem MH,
van Gorp EC. Derangements of the coagulation system in patients with severe leptospirosis is associated with
severe bleeding and mortality. Tropical Medicine and International Health, in press
Gonçalves AT, Paiva C, Melo-Mota F, Vieira ML, Carreira T, Nunes MS, Mota-Vieira L, Ahmed A, Hartskeerl
RA, Hyde K and Collares-Pereira M. First isolation of human Leptospira strains (Azores, Portugal). International
Journal of Infectious Diseases, in press.
Abela-Ridder B, Sikkema R, Hartskeerl RA. Estimating the burden of human leptospirosis. International Journal
of Antimicrobial Agents, in press.
4 Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
13. Inscription of diagnostic kits on the OIE Register
i) Did you participate in expert panels for the validation of candidate kits for inscription on the
OIE Register? If yes, for which kits?
ii) Did you submit to the OIE candidate kits for inscription on the OIE Register? If yes, for
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009 5