Rudy A. Hartskeerl

Comparative Genomics of Two Leptospira interrogans Serovars Reveals Novel Insights into Physiology and Pathogenesis

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide O side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organisms complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

Detection of seven species of pathogenic leptospires by PCR using two sets of primers

Two sets of primers derived from genomic DNA libraries of Leptospira serovars icterohaemorrhagiae (strain RGA) and bim (strain 1051) enabled the amplification by PCR of target DNA fragments from leptospiral reference strains belonging to all presently described pathogenic Leptospira species. The icterohaemorrhagiae-derived primers (G1/G2) enabled amplification of DNA from L. interrogans, L. borgpetersenii, L. weilii, L. noguchii, L. santarosai and L. meyeri, whereas the bim-derived primers (B64-I/B64-II) enabled the amplification of L. kirschneri. Southern blot and DNA sequence analysis revealed inter-species DNA polymorphism within the region spanned by primers G1 and G2 between L. interrogans and various other Leptospira species. Using a mixture of primer sets G1/G2 and B64-I/B64-II, leptospires of serovars icterohaemorrhagiae, copenhageni, hardjo, pomona, grippotyphosa and bim were detected in serum samples collected from patients during the first 10 days after the onset of illness.

Multilocus sequence typing method for identification and genotypic classification of pathogenic Leptospira species

BackgroundLeptospira are the parasitic bacterial organisms associated with a broad range of mammalian hosts and are responsible for severe cases of human Leptospirosis. The epidemiology of leptospirosis is complex and dynamic. Multiple serovars have been identified, each adapted to one or more animal hosts. Adaptation is a dynamic process that changes the spatial and temporal distribution of serovars and clinical manifestations in different hosts. Serotyping based on repertoire of surface antigens is an ambiguous and artificial system of classification of leptospiral agents. Molecular typing methods for the identification of pathogenic leptospires up to individual genome species level have been highly sought after since the decipherment of whole genome sequences. Only a few resources exist for microbial genotypic data based on individual techniques such as Multiple Locus Sequence Typing (MLST), but unfortunately no such databases are existent for leptospires.ResultsWe 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, icd A and sec Y were significantly more variable whereas the LipL32 and LipL41 coding genes and the rrs 2 gene were moderately variable. The phylogenetic tree clustered the isolates according to the genome-based species.ConclusionThe 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.

Development and Validation of a Real-Time PCR for Detection of Pathogenic Leptospira Species in Clinical Materials

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 and immunological characterization of the microsporidian Septata intestinalis Cali, Kotler and Orenstein, 1993: reclassification to Encephalitozoon intestinalis.

The relationships between the Encephalitozoon-like Septata intestinalis and other microsporidia that occur in humans; notably Encephalitozoon cuniculi and Encephalitozoon hellem, is insufficiently documented using morphological descriptions alone. To assess mutual relationships, we have examined other phenotypic as well as genetic aspects of S. intestinalis, obtained both from tissue culture and clinical specimens, in comparison with a number of other microsporidia. Phenotypic characterization was performed by analysis of the protein composition and antigenic structure of various microsporidian spores by SDS-PAGE and Western blotting. The genetic characterization consisted of the determination of the sequence of the S. intestinalis rrs gene encoding the small subunit ribosomal RNA (srRNA), restriction fragment length polymorphism (RFLP) analysis of amplified rrs genes and establishment of the degree of sequence identity between rrs genes of various microsporidian species. The unique sequence of rrs of S. intestinalis as well as the distinct RFLP and SDS-PAGE profiles indicate that S. intestinalis is clearly different from other human microsporidian species. However, its rrs gene shared about 90% sequence identity with rrs of both Encephalitozoon spp., E. cuniculi and E. hellem. This is remarkably higher than the about 70% identity observed between rrs of microsporidian species which belong to different genera and thus suggests that S. intestinalis should be regarded as a species of the genus Encephalitozoon.(ABSTRACT TRUNCATED AT 250 WORDS)

Leptospirosis, Water Sports, and Chemoprophylaxis

Recreational activities, such as water sports and adventure travel, are emerging as an important risk factor for leptospirosis, a potentially fatal zoonosis. We report the clinical course of 2 patients who acquired leptospirosis through participation in water sports. Physicians caring for patients who participate in adventure travel involving water sports should be familiar with the risk factors for and diagnosis, prevention, and treatment of leptospirosis.

Polymerase Chain Reaction for the Detection of Mycobacterium leprae

A polymerase chain reaction (PCR) using heat-stable Taq polymerase is described for the specific detection of Mycobacterium leprae, the causative agent of leprosy. A set of primers was selected on the basis of the nucleotide sequence of a gene encoding the 36 kDa antigen of M. leprae. With this set of primers in the PCR, M. leprae could be detected specifically with a detection limit approximating one bacterium. This PCR appears to meet the criteria of specificity and sensitivity required for a useful tool in epidemiology and eventually for the control of leprosy.

Carriage of Leptospira interrogans among domestic rats from an urban setting highly endemic for leptospirosis in Brazil

A survey was conducted to identify reservoirs for urban leptospirosis in the city of Salvador, Brazil. Sampling protocols were performed in the vicinity of households of severe leptospirosis cases identified during active hospital-based surveillance. Among a total of 142 captured Rattus norvegicus (Norwegian brown rat), 80.3% had a positive culture isolate from urine or kidney specimens and 68.1% had a positive serum sample by microscopic agglutination test (MAT) titre of > or = 1:100. Monoclonal antibody-based typing of isolates identified that the agent carried by rats was Leptospira interrogans serovar Copenhageni, which was the same serovar isolated from patients during hospital-based surveillance. Leptospira spp. were not isolated from 8 captured Didelphis marsupialis (Opossum), while 5/7 had a positive MAT titre against a saprophytic serogroup. R. rattus were not captured during the survey. The study findings indicate that the brown rat is a major rodent reservoir for leptospirosis in this urban setting. Furthermore, the high carriage rates of L. interrogans serovar Copenhageni in captured rats suggest that there is a significant degree of environmental contamination with this agent in the household environment of high risk areas, which in turn is a cause of transmission during urban epidemics.

Characterization of Encephalitozoon (Septata) intestinalis isolates cultured from nasal mucosa and bronchoalveolar lavage fluids of two AIDS patients

ABSTRACT. Microsporidia are obligate intracellular protozoan parasites that can cause opportunistic infections in AIDS patients. Species from five genera of microsporidia are presently known to infect man. One species, Septata intestinalis originally was detected in stool specimens of individuals with chronic diarrhea and subsequently was found to disseminate to the kidneys, lungs, and nasal sinuses. This organism has since been reclassified as Encephalitozoon and in this study, we report the culture of Encephalitozoon intestinalis from a bronchoalveolar lavage specimen and a nasal mucus aspirate of two AIDS patients living in the USA. The bronchoalveolar and nasal microsporidian isolates grew in several continuous cell lines including RK‐13, MDCK, HT‐29, Caco‐2, Vero, and 1047. Transmission electron microscopy of the clinical and cell culture specimens revealed that the new isolates appeared to be E. intestinalis based on morphology and growth of organisms in septated membrane‐bound parasitophorous vacuoles. The new E. intestinalis isolates were characterized and compared with the first isolated E. intestinalis that was cultured from stool to confirm their identity and to determine if there existed any minor differences, as seen in the closely related Encephalitozoon cuniculi strains. By the methods of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis staining for proteins and carbohydrates, Western blot immunodetection, and polymerase chain reaction‐based methods with restriction endonuclease digestion, double‐stranded DNA heteroduplex mobility shift analysis, and DNA sequencing of the ribosomal DNA intergenic spacer region, the new isolates were identical to each other and to the reference isolate of E. intestinalis. In addition, with any of these methods, the E. intestinalis organisms could be distinguished from the three E. cuniculi strains, Encephalitozoon hellem, and Vittaforma corneae, which is important for diagnostics, therapeutic strategies, and epidemiology.

Estimating the burden of human leptospirosis

Leptospirosis, a disease that is often under- or misdiagnosed, significantly impacts human health in many parts of the world and generally affects the most vulnerable communities. Obtaining reliable and comparable information about the occurrence of leptospirosis in populations, and detecting changing trends, are critical for setting policy and public health priorities. Traditional sources of information about the descriptive epidemiology, the disability attributed to leptospirosis infection, and associated risk factors are generally incomplete, fragmented and of uncertain reliability and comparability. Therefore, the global burden of disease concept and methodological framework will be used by the World Health Organizations (WHO) initiative to estimate the global burden of human leptospirosis. The aim of the initiative is to quantify and compare the health of populations by a summary measure of both mortality and disability, the disability-adjusted life year (DALY). WHO has established the Leptospirosis Burden Epidemiology Reference Group (LERG) to coordinate the assessment. The burden estimates provided by the LERG will guide public health policy on leptospirosis disease control and prevention, with the aim of reducing the impact on human health.