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Search Results: 1 - 10 of 462184 matches for " Rudy A. Hartskeerl "
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International Leptospirosis Society: objectives and achievements Sociedad Internacional de Leptospirosis: objetivos y realización
Rudy A. Hartskeerl
Revista Cubana de Medicina Tropical , 2005,
Evaluation of Real-Time PCR and Culturing for the Detection of Leptospires in Canine Samples  [PDF]
Ahmed Ahmed, Henricus L. B. M. Klaasen, Mark van der Veen, Hans van der Linden, Marga G. A. Goris, Rudy A. Hartskeerl
Advances in Microbiology (AiM) , 2012, DOI: 10.4236/aim.2012.22021
Abstract: Validated real-time PCRs detecting leptospires for veterinary purposes are not readily available. This paper describes the prospective evaluation of a SYBR Green-based real-time PCR on serum samples collected from experimentally infected dogs. Compared to culturing, the assay had a diagnostic sensitivity and specificity of 91.7% and 90.0%, respectively. Culturing for part is complementary to PCR and preferably both should be applied for diagnosis and vaccine challenge experiments. In a subsequent prospective study on the dynamics of experimental infections with serovars Canicola and Copenhageni and serovar complex Bananal-Liangguang in young and adult dogs, the PCR was applied on serum samples in conjunction with culturing on blood, urine and kidney samples with the following results: 1) Leptospires persisted longer in the blood of young dogs than of adult ones; 2) Numbers of viable leptospires in the blood are rapidly reduced but DNA remains occasionally detectable up to 7 days post infection; 3) Appearance of viable leptospires in the urine follows a biphasic dynamics; 4) EDTA hampers effective culturing from blood samples; 5) Serovar Canicola persists longer in the blood of dogs than Copenhageni and Bananal-Liangguang. Together with a markedly higher recovery rate from kidney samples (71% compared to respectively 33% and 0% in young dogs), this probably reflects the adaptation power of Canicola to its canine maintenance host. Appearance of leptospires in the urine samples indicates that experimental infections have been successful. PCR presents a valuable adjunct to the diagnosis of veterinary leptospirosis and the follow-up in vaccine protection experiments.
Development and Validation of a Real-Time PCR for Detection of Pathogenic Leptospira Species in Clinical Materials
Ahmed Ahmed, Mirjam F. M. Engelberts, Kimberly R. Boer, Niyaz Ahmed, Rudy A. Hartskeerl
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0007093
Abstract: Available serological diagnostics do not allow the confirmation of clinically suspected leptospirosis at the early acute phase of illness. Several conventional and real-time PCRs for the early diagnosis of leptospirosis have been described but these have been incompletely evaluated. We developed a SYBR Green-based real-time PCR targeting secY and validated it according to international guidelines. To determine the analytical specificity, DNA from 56 Leptospira strains belonging to pathogenic, non-pathogenic and intermediate Leptospira spp. as well as 46 other micro-organisms was included in this study. All the pathogenic Leptospira gave a positive reaction. We found no cross-reaction with saprophytic Leptospira and other micro-organisms, implying a high analytical specificity. The analytical sensitivity of the PCR was one copy per reaction from cultured homologous strain M 20 and 1.2 and 1.5 copy for heterologous strains 1342 K and Sarmin, respectively. In spiked serum & blood and kidney tissue the sensitivity was 10 and 20 copies for M 20, 15 and 30 copies for 1342 K and 30 and 50 copies for Sarmin. To determine the diagnostic sensitivity (DSe) and specificity (DSp), clinical blood samples from 26 laboratory-confirmed and 107 negative patients suspected of leptospirosis were enrolled as a prospective consecutive cohort. Based on culture as the gold standard, we found a DSe and DSp of 100% and 93%, respectively. All eight PCR positive samples that had a negative culture seroconverted later on, implying a higher actual DSp. When using culture and serology as the gold standard, the DSe was lower (89%) while the DSp was higher (100%). DSe was 100% in samples collected within the first – for treatment important - 4 days after onset of the illness. Reproducibility and repeatability of the assay, determined by blind testing kidney samples from 20 confirmed positive and 20 negative rodents both appeared 100%. In conclusion we have described for the first time the development of a robust SYBR Green real-time PCR for the detection of pathogenic Leptospira combined with a detailed assessment of its clinical accuracy, thus providing a method for the early diagnosis of leptospirosis with a well-defined satisfactory performance.
Genetic Affinities within a Large Global Collection of Pathogenic Leptospira: Implications for Strain Identification and Molecular Epidemiology
Kishore Nalam,Ahmed Ahmed,Sundru Manjulata Devi,Paolo Francalacci,Mumtaz Baig,Leonardo A. Sechi,Rudy A. Hartskeerl,Niyaz Ahmed
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0012637
Abstract: Leptospirosis is an important zoonosis with widespread human health implications. The non-availability of accurate identification methods for the individualization of different Leptospira for outbreak investigations poses bountiful problems in the disease control arena. We harnessed fluorescent amplified fragment length polymorphism analysis (FAFLP) for Leptospira and investigated its utility in establishing genetic relationships among 271 isolates in the context of species level assignments of our global collection of isolates and strains obtained from a diverse array of hosts. In addition, this method was compared to an in-house multilocus sequence typing (MLST) method based on polymorphisms in three housekeeping genes, the rrs locus and two envelope proteins. Phylogenetic relationships were deduced based on bifurcating Neighbor-joining trees as well as median joining network analyses integrating both the FAFLP data and MLST based haplotypes. The phylogenetic relationships were also reproduced through Bayesian analysis of the multilocus sequence polymorphisms. We found FAFLP to be an important method for outbreak investigation and for clustering of isolates based on their geographical descent rather than by genome species types. The FAFLP method was, however, not able to convey much taxonomical utility sufficient to replace the highly tedious serotyping procedures in vogue. MLST, on the other hand, was found to be highly robust and efficient in identifying ancestral relationships and segregating the outbreak associated strains or otherwise according to their genome species status and, therefore, could unambiguously be applied for investigating phylogenetics of Leptospira in the context of taxonomy as well as gene flow. For instance, MLST was more efficient, as compared to FAFLP method, in clustering strains from the Andaman island of India, with their counterparts from mainland India and Sri Lanka, implying that such strains share genetic relationships and that leptospiral strains might be frequently circulating between the islands and the mainland.
Conservation of the S10-spc-α Locus within Otherwise Highly Plastic Genomes Provides Phylogenetic Insight into the Genus Leptospira
Berta Victoria, Ahmed Ahmed, Richard L. Zuerner, Niyaz Ahmed, Dieter M. Bulach, Javier Quinteiro, Rudy A. Hartskeerl
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0002752
Abstract: S10-spc-α is a 17.5 kb cluster of 32 genes encoding ribosomal proteins. This locus has an unusual composition and organization in Leptospira interrogans. We demonstrate the highly conserved nature of this region among diverse Leptospira and show its utility as a phylogenetically informative region. Comparative analyses were performed by PCR using primer sets covering the whole locus. Correctly sized fragments were obtained by PCR from all L. interrogans strains tested for each primer set indicating that this locus is well conserved in this species. Few differences were detected in amplification profiles between different pathogenic species, indicating that the S10-spc-α locus is conserved among pathogenic Leptospira. In contrast, PCR analysis of this locus using DNA from saprophytic Leptospira species and species with an intermediate pathogenic capacity generated varied results. Sequence alignment of the S10-spc-α locus from two pathogenic species, L. interrogans and L. borgpetersenii, with the corresponding locus from the saprophyte L. biflexa serovar Patoc showed that genetic organization of this locus is well conserved within Leptospira. Multilocus sequence typing (MLST) of four conserved regions resulted in the construction of well-defined phylogenetic trees that help resolve questions about the interrelationships of pathogenic Leptospira. Based on the results of secY sequence analysis, we found that reliable species identification of pathogenic Leptospira is possible by comparative analysis of a 245 bp region commonly used as a target for diagnostic PCR for leptospirosis. Comparative analysis of Leptospira strains revealed that strain H6 previously classified as L. inadai actually belongs to the pathogenic species L. interrogans and that L. meyeri strain ICF phylogenetically co-localized with the pathogenic clusters. These findings demonstrate that the S10-spc-α locus is highly conserved throughout the genus and may be more useful in comparing evolution of the genus than loci studied previously.
Bioinformatics Describes Novel Loci for High Resolution Discrimination of Leptospira Isolates
Gustavo M. Cerqueira,Alan J. A. McBride,Rudy A. Hartskeerl,Niyaz Ahmed,Odir A. Dellagostin,Marcus R. Eslab?o,Ana L. T. O. Nascimento
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015335
Abstract: Leptospirosis is one of the most widespread zoonoses in the world and with over 260 pathogenic serovars there is an urgent need for a molecular system of classification. The development of multilocus sequence typing (MLST) schemes for Leptospira spp. is addressing this issue. The aim of this study was to identify loci with potential to enhance Leptospira strain discrimination by sequencing-based methods.
Multilocus sequence typing method for identification and genotypic classification of pathogenic Leptospira species
Niyaz Ahmed, S Manjulata Devi, M de los á Valverde, P Vijayachari, Robert S Machang'u, William A Ellis, Rudy A Hartskeerl
Annals of Clinical Microbiology and Antimicrobials , 2006, DOI: 10.1186/1476-0711-5-28
Abstract: We for the first time report development of a robust MLST method for genotyping of Leptospira. Genotyping based on DNA sequence identity of 4 housekeeping genes and 2 candidate genes was analyzed in a set of 120 strains including 41 reference strains representing different geographical areas and from different sources. Of the six selected genes, adk, icdA and secY were significantly more variable whereas the LipL32 and LipL41 coding genes and the rrs2 gene were moderately variable. The phylogenetic tree clustered the isolates according to the genome-based species.The main advantages of MLST over other typing methods for leptospires include reproducibility, robustness, consistency and portability. The genetic relatedness of the leptospires can be better studied by the MLST approach and can be used for molecular epidemiological and evolutionary studies and population genetics.Leptospirosis is a zoonotic and an emerging infectious disease caused by the pathogenic Leptospira species and is identified in the recent years as a global public health problem because of its increased mortality and morbidity in different countries. Leptospirosis is frequently misdiagnosed as a result of its protean and non-specific presentation resembling many other febrile diseases, notably viral haemorrhagic fevers such as dengue [1]. There is, for certain, an underestimation of the leptospirosis problem due to lack of awareness and under-recognition through a lack of proper use of diagnostic tools.The common mode of transmission of the infection in humans is either by direct or indirect contact with the urine of infected animals and may lead to potential lethal disease. A unique feature of this organism is to parasitize in a wide variety of wild and domestic animals [2]. Traditionally, two species have been identified, i.e. Leptospira interrogans and L. biflexa for pathogenic and non-pathogenic leptospires, respectively. The serovar is the basic identifier, characterized on the basis of ser
Prospective Evaluation of Three Rapid Diagnostic Tests for Diagnosis of Human Leptospirosis
Marga G. A. Goris ,Mariska M. G. Leeflang,Martin Loden,Jiri F. P. Wagenaar,Paul R. Klatser,Rudy A. Hartskeerl,Kimberly R. Boer
PLOS Neglected Tropical Diseases , 2013, DOI: 10.1371/journal.pntd.0002290
Abstract: Background Diagnosis of leptospirosis by the microscopic agglutination test (MAT) or by culture is confined to specialized laboratories. Although ELISA techniques are more common, they still require laboratory facilities. Rapid Diagnostic Tests (RDTs) can be used for easy point-of-care diagnosis. This study aims to evaluate the diagnostic performance of the RDTs LeptoTek Dri Dot, LeptoTek Lateral Flow, and Leptocheck-WB, prospectively. Methodology During 2001 to 2012, one or two of the RDTs at the same time have been applied prior to routine diagnostics (MAT, ELISA and culture) on serum specimens from participants sent in for leptospirosis diagnosis. The case definition was based on MAT, ELISA and culture results. Participants not fulfilling the case definition were considered not to have leptospirosis. The diagnostic accuracy was determined based on the 1st submitted sample and paired samples, either in an overall analysis or stratified according to days post onset of illness. Results The overall sensitivity and specificity for the LeptoTek Dri Dot was 75% respectively 96%, for the LeptoTek Lateral Flow 78% respectively 95%, and for the Leptocheck-WB 78% respectively 98%. Based on the 1st submitted sample the sensitivity was low (51% for LeptoTek Dri Dot, 69% for LeptoTek Lateral Flow, and 55% for Leptocheck-WB), but substantially increased when the results of paired samples were combined, although accompanied by a lower specificity (82% respectively 91% for LeptoTek Dri Dot, 86% respectively 84% for LeptoTek Lateral Flow, and 80% respectively 93% for Leptocheck-WB). Conclusions All three tests present antibody tests contributing to the diagnosis of leptospirosis, thus supporting clinical suspicion and contributing to awareness. Since the overall sensitivity of the tested RDTs did not exceed 80%, one should be cautious to rely only on an RDT result, and confirmation by reference tests is strongly recommended.
Towards the Burden of Human Leptospirosis: Duration of Acute Illness and Occurrence of Post-Leptospirosis Symptoms of Patients in The Netherlands
Marga G. A. Goris, Vanessa Kikken, Masja Straetemans, Sandra Alba, Marco Goeijenbier, Eric C. M. van Gorp, Kimberly R. Boer, Jiri F. P. Wagenaar, Rudy A. Hartskeerl
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0076549
Abstract: Background Leptospirosis is a global zoonotic disease. Although important for the assessment of the burden of leptospirosis, data on the duration of the illness and the occurrence of post-leptospirosis complaints are not well documented. Hence the main objective of this study was to estimate the occurrence of persistent complaints and duration of hospital stay in laboratory confirmed leptospirosis patients in the Netherlands during 1985 to 2010. Additionally, several risk factors potentially impacting on the occurrence of post-leptospirosis complaints were investigated. Methods/Principal Findings The duration of the acute phase of leptospirosis was 16 days (IQR 12–23); 10 days (IQR 7–16) were spent hospitalized. Eighteen fatal cases were excluded from this analysis. Complaints of leptospirosis patients by passive case investigations (CPC) derived from files on ambulant consultations occurring one month after hospital discharge, revealed persistent complaints in 108 of 236 (45.8%) laboratory confirmed cases. Data on persistent complaints after acute leptospirosis (PCAC), assessed in 225 laboratory confirmed leptospirosis cases collected through questionnaires during 1985-1993, indicated 68 (30.2%) PCAC cases. Frequently reported complaints included (extreme) fatigue, myalgia, malaise, headache, and a weak physical condition. These complaints prolonged in 21.1% of the cases beyond 24 months after onset of disease. There was no association between post-leptospirosis complaints and hospitalization. However, individuals admitted at the intensive care unit (ICU) were twice as likely to have continuing complaints after discharge adjusting for age and dialysis (OR 2.0 95% CI 0.8-4.8). No significant association could be found between prolongation of complaints and infecting serogroup, although subgroup analysis suggest that infection with serogroups Sejroe (OR 4.8, 95%CI 0.9-27.0) and icterohaemorrhagiae (OR 2.0, 95%CI 0.9-4.3 CI) are more likely to result in CPC than infections with serogroup Grippotyphosa. Conclusion/Significance In addition to the acute disease, persistent complaints have an impact on the burden of leptospirosis.
Potent Innate Immune Response to Pathogenic Leptospira in Human Whole Blood
Marga G. A. Goris,Jiri F. P. Wagenaar,Rudy A. Hartskeerl,Eric C. M. van Gorp,Simone Schuller,Avril M. Monahan,Jarlath E. Nally,Tom van der Poll,Cornelis van 't Veer
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0018279
Abstract: Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. The bacteria enter the human body via abraded skin or mucous membranes and may disseminate throughout. In general the clinical picture is mild but some patients develop rapidly progressive, severe disease with a high case fatality rate. Not much is known about the innate immune response to leptospires during haematogenous dissemination. Previous work showed that a human THP-1 cell line recognized heat-killed leptospires and leptospiral LPS through TLR2 instead of TLR4. The LPS of virulent leptospires displayed a lower potency to trigger TNF production by THP-1 cells compared to LPS of non-virulent leptospires.
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