A polymerase chain reaction and single-strand conformation polymorphism determination (PCR-SSCP) was used to detect deoxyribonucleic acid sequence polymorphisms in the transcribed non-coding regions between the small and large sub-unit ribosomal ribonucleic acid (rRNA) genes in Leishmania donovani from 63 clinical samples collected in eastern Sudan, between April 1997 and October 1998. Specific Leishmania primers were used to amplify the internal transcribed spacer (ITS) regions of L. donovani isolates directly from clinical samples spotted on filter papers. Amplification products were subsequently analysed by SSCP. Eleven polymorphic patterns were detected in the first part of the spacer, the ITS1 region, and were sequenced. Most of the changes were due to deletions of adenine bases and AT pairs within the first 192 nucleotides of the ITS region. This is the first application of PCR-linked SSCP analysis for the detection of population variation with direct display of sequence variation in parasitologically positive clinical samples spotted on filter paper. Culturing the parasite is thus not required, which is beneficial particularly in epidemiological studies based on field work where obtaining cultures can be extremely difficult.

Animals↗

PubMedDate not supplied

Genetic heterogeneity in the species Leishmania tropica revealed by different PCR-based methods.

A PCR fingerprinting approach, using single non-specific primers, as well as restriction and single-strand conformation polymorphism (SSCP) analyses of the amplified ribosomal internal transcribed spacer, were used to investigate genetic variability in the species Leishmania tropica. Twenty-nine strains of the 'L. tropica complex' from different Old World geographical areas were studied including 4 from Namibia, and 1 strain of L. killicki. All techniques revealed a high degree of genetic heterogeneity among the strains of L. tropica. The PCR fingerprinting displayed the highest discriminatory power, but can be applied only to cultured parasites. The internal transcribed spacer (ITS) region can be amplified directly from infected clinical samples and analysed subsequently. No strict correlation was discerned between the genetic variants and either the geographical origin of the strains or the clinical manifestations associated with human disease, except for the Namibian strains. Also, genetic variation did not correlate well with characterization by enzyme variant electrophoretic analysis. The strain of L. killicki always clustered together with the strains of L. tropica, suggesting it, probably, should not be considered a separate species of Leishmania. However, the 4 Namibian strains formed a distinct, statistically well-supported group closely related to but different from the other strains of L. tropica.

Animals↗

PubMedDate not supplied

Identification of clinical strains of Candida albicans by DNA fingerprinting with the polymerase chain reaction.

DNA polymorphisms generated by the polymerase chain reaction (PCR) were used to differentiate clinical isolates of Candida. This PCR method employed single primers that were originally designed as hybridization probes for DNA fingerprinting experiments to probe minisatellite and microsatellite DNA sequences. To evaluate this procedure, 35 isolates from 20 patients in several intensive care units and 12 isolates obtained from the oral cavities of healthy dental patients were fingerprinted. The PCR-fingerprint patterns of isolates of Candida albicans from the immunocompromised patients revealed fewer differences than isolates from the dental service. Multiple isolates from different body sites of the same patients revealed that patients may harbour isolates of Candida with the same or different PCR-fingerprints. Since this method is generally simpler and faster than established methods of biotyping medically important yeasts, PCR-fingerprinting may prove useful for the surveying of large numbers of pathogens for epidemiological studies.

Base Sequence↗

PubMedDate not supplied

Toxoplasma infection and cell free extract of the parasites are able to reverse multidrug resistance of mouse lymphoma and human gastric cancer cells in vitro.

A large number of compounds are known to reduce the ATP-dependent efflux pump activity of multidrug resistant (mdr) tumor cells. Here we report that an infection of cancer cells with T. gondii reduced the multidrug resistance of the tumour cells against cytostatic drugs. Two mouse lymphoma cell lines (Mdr L 5718 and Par 5718) were infected with Toxoplasma gondii in vitro and the reduction of efflux pump activity of the cells was measured. The drug accumulation (Rhodamin-123) was increased in the infected mdr cell lines compared with non- infected mdr-cells, and no effect was shown after infection of the parental cell line. The same effect was also achieved by incubation of Mdr-tumor cells with cell lysate of Toxoplasma gondii. Mdr-1-gene expression was reduced in the infected cell lines 48 hours after infection. Co-cultivation of Toxoplasma gondii with mdr cell lines separated by a microfilter from tumor cells was performed, but this cocultivation did not change the mdr efflux activity. The effect of Toxoplasma gondii infection on the efflux pump activity and mdr-1 gene expression was also examined in the human gastric cancer cells. A sensitization of resistant gastric cancer cells was also achieved by parasite infection. This phenomenon is an evidence that a reduction of resistance in tumor cells can be achieved by a natural parasite infection. It is as yet unclear whether an active infection or another substance of T. gondii is responsible for this phenomenon.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMedDate not supplied

[An edited segment of the kinetoplast gene MURF4 (ATPase 6) in leishmania has a similar structure].

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Adenosine Triphosphatases↗