W. J. Terpstra

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.

ELISA for the Detection of Specific IgM and IgG in Human Leptospirosis

ELISA was used to detect specific IgM and IgG in sera from humans with current or past leptospirosis. A serological pattern of a high IgM titre (greater than or equal to 1280), or moderately increased IgM (160-640) in conjunction with a low IgG titre (less than or equal to 20), with serovar copenhageni antigen was characteristic for approximately two-thirds of the sera from serovar icterohaemorrhagiae patients obtained in the first two months of the disease. The antigen was the supernatant of a heated and centrifuged culture of leptospires. Antigens were prepared from serovars copenhageni, grippotyphosa, hardjo and patoc. Sera from patients with icterohaemorrhagiae, grippotyphosa and hardjo infections showed cross-reactivity when different antigens were used. In past infections the IgG titres were clearly higher with the homologous antigen. ELISA for IgM and IgG allows the rapid diagnosis of acute leptospirosis.

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)

Clinical significance of small-intestinal microsporidiosis in HIV-1-infected individuals

To assess the importance of microsporidiosis of the small intestine in the pathogenesis of chronic diarrhoea in HIV-1-infected individuals, duodenal biopsy samples from the following three patient groups were prospectively evaluated for bacterial, viral, and parasitic pathogens by standard methods, and for microsporidia by light microscopy: 55 consecutive HIV-1-antibody-positive subjects with unexplained diarrhoea of at least 3 weeks duration (group A); 38 HIV-1-seropositive subjects without diarrhoea (group B) who consecutively underwent upper gastrointestinal endoscopy for various reasons; and 7 patients without known risk factors for HIV infection with chronic unexplained diarrhoea (group C). In groups A and B most subjects had had previous AIDS-defining opportunistic infections and the median peripheral blood CD4 lymphocyte count was less than 0.1 x 10(9)/l. Microsporidia were detected as the single pathogen in 15 of the group A compared with 1 (in whom diarrhoea subsequently developed) of the group B patients (p = 0.001) and none of the group C patients. With the exception of 4 of the group A patients, no other intestinal pathogens were identified in any of the patients. The median peripheral blood CD4 count was significantly lower in patients with detectable microsporidia than in those without microsporidiosis (0.03 x 10(9)/l vs 0.06 x 10(9)/l; p = 0.03); in all patients with microsporidiosis, the CD4 count was equal to or less than 0.1 x 10(9)/l. 13 patients with microsporidiosis were treated with metronidazole, in 10 of whom treatment led to a substantial improvement or disappearance of diarrhoea within days of starting therapy, but did not result in eradication of the parasite in the 5 patients who underwent repeat biopsy. The findings suggest that small-intestinal microsporidiosis is an important cause of chronic unexplained diarrhoea in HIV-1-infected individuals with pronounced cellular immune deficiency. This infection should therefore be added to the list of AIDS-defining opportunistic infections.

Serodiagnosis of Human Leptospirosis by Enzyme-Linked-Immunosorbent-Assay (ELISA)

An enzyme-linked-immunosorbent-assay (ELISA) is described for the serodiagnosis of leptospirosis. Using an antigen prepared from a heated culture of a single leptospira strain (Wijnberg) the ELISA is a genusspecific test. The microscopic agglutination test (MAT) served as a reference. ELISA and MAT results agreed in 95% of the sera from 96 leptospirosis patients. One false positive was found in 217 controls. The ELISA is sensitive, specific and relatively easy to perform.

Identification of 'Old World' Leishmania by DNA recombinant probes.

Leishmania are usually identified by iso-enzyme analysis. This method works well, but there is a need for an additional, more simple, method of identification. Here we present data that show that in a Southern blot analysis, recombinant DNA probes in combination with certain restriction enzymes can differentiate between taxa of Leishmania. Probes based on clones selected from a L. infantum cDNA library gave characteristic patterns on Southern blots for reference strains of the different types of Leishmania found in Europe, Africa and Asia. Within the different taxa little or no variation was observed. Although the L. infantum derived probes showed a somewhat stronger hybridization for strains of the L. donovani complex, the signal obtained with most probes was satisfactory for L. major, L. aethiopica and L. tropica. Within the L. donovani complex none of the selected probes differentiated between isolates belonging to L. infantum, L. chagasi or L. donovani. Probes containing kinetoplast DNA showed considerable variation in hybridization within a taxon.

The Classification of Sejroe Group Serovars of Leptospira interrogans with Monoclonal Antibodies

Using the hybridoma technique we produced monoclonal antibodies to serovars of Leptospira interrogans. We focussed on serovar hardjo which is an important pathogen for humans and animals, and on other serovars of the Sejroe group. With combinations of monoclonals, characteristic patterns of agglutination were observed according to the specific pattern of antigenic determinants of that serovar. A set of 13 monoclonal antibodies was composed which allowed the classification of almost all serovars of the Sejroe group.

Detection of leptospiral DNA by nucleic acid hybridisation with 32P- and biotin-labelled probes.

Dot hybridisation with 32P- and biotin-labelled probes prepared from leptospiral DNA was performed to develop a sensitive and specific diagnostic method for early infection with leptospires. The smallest amounts of leptospiral DNA that could be detected with 32P- and biotin-labelled probes was 1.5 pg and 5 pg, respectively, corresponding to about 750 and 2500 leptospires. Dot hybridisation with a 32P-labelled probe detected leptospiral DNA in sera from all of 14 experimentally infected golden hamsters. The smallest amount of leptospiral DNA detected in these experiments corresponded to about 2500 leptospires. In the test conditions described in this study, the sensitivity of dot hybridisation with a biotin-labelled probe was lower. Little cross-hybridisation was observed with unrelated DNAs which indicates that dot hybridisation could be a useful diagnostic method.

Direct detection of leptospiral material in human postmortem samples

Leptospiral culture, direct immunofluorescence, and the polymerase chain reaction (PCR) were used to detect leptospiral material in postmortem specimens collected from eight patients who died of leptospirosis. Diagnosis of leptospiral infection was based on clinical summary (premortem) and confirmed by serological analysis and/or culture of leptospires. Leptospiral culture was the least sensitive technique, yielding two isolates (3%) from 65 samples. Both isolates were from the aqueous humour and cerebrospinal fluid of the same patient. Direct immunofluorescence was of intermediate sensitivity for detection of leptospires, confirming the presence of leptospires in 11% (2 of 18) of tissue samples from three patients. PCR analysis was the most sensitive technique for detection of leptospiral material in tissue samples, being positive in 20% (11 of 56) of samples from eight patients. Both samples (cerebellum and liver) positive by immunofluorescence were also positive by PCR. The sensitivity of the PCR assay was 1-10 leptospires ml(-1) sample, and the assay was specific for Leptospira pathogenic species. Multi-system involvement was indicated based on successful amplification of leptospiral DNA from more than one tissue sample, which corroborated with the clinical and pathologic findings. The results suggest that in acute and/or fatal leptospirosis, the pathogenesis of the pathologic features are related to the presence of the organisms in the tissues. In conclusion, PCR combined with serology appears to be a useful tool for diagnosis of leptospirosis and may be invaluable in epidemiological studies.

DNA hybridization with hardjobovis-specific recombinant probes as a method for type discrimination of Leptospira interrogans serovar hardjo.

Restriction endonuclease analysis of DNA of Leptospira interrogans, serovar hardjo, showed two distinct types within this serovar. These two types, hardjoprajitno and hardjobovis, cannot be differentiated by monoclonal antibodies. Application of 32P- or biotin-labelled total DNA probes in dot-blot or in situ hybridization assays showed a high sensitivity of the assays but also considerable cross-hybridization. Therefore, a genomic library of hardjobovis was constructed and a number of hardjobovis-specific recombinant clones were isolated. Finally, four clones were selected on the basis of a strong hybridization signal and a high specificity for hardjobovis as compared to hardjoprajitno. In a dot-blot assay as well as in in situ hybridization experiments all four clones gave strong signals, and no cross-hybridization with hardjoprajitno was observed in either type of assay. Our results indicate that specific recombinant DNA probes might provide tools for routine diagnosis and classification in cases of hardjo infections.