Biomedical subjects
Publications and source records attributed to A J Simpson.
Pleural infection with Clostridium difficile is extremely rare. A case of nosocomial empyema following chest drain insertion in a 46 year old man is described. The potential of C difficile to cause extra-intestinal infections should be recognised and its isolation from other sites should not be ignored.
A case of brain abscess involving Haemophilus paraphrophilus and Actinomyces odontolyticus is presented. This combination of organisms has not previously been described. All brain abscess specimens should routinely be processed rapidly and cultured for a prolonged period to ensure recovery of fastidious organisms which may have implications for antibiotic therapy.
We have developed a technique called "LSSP-PCR" (low-stringency single specific primer PCR) that detects single or multiple mutations in DNA. A purified DNA fragment is submitted to PCR by using a single primer specific for one of the extremities of the fragment, under conditions of very low stringency. The primer hybridizes specifically to its complementary extremity and nonspecifically to multiple sites within the fragment, in a sequence-dependent manner. A complex set of reaction products is thus created that, when separated by electrophoresis, constitutes a unique "gene signature." We here report the application of LSSP-PCR to the detection of sequence variation in the control (D-loop) region of human mtDNA, which is known to differ significantly between unrelated individuals. We prepared human DNA samples from blood and amplified a 1024-bp portion of the mtDNA control region, using primers L15996 and H408. The amplified mtDNA fragments were then reamplified under LSSP-PCR conditions by using L15996 or H408 as drivers to produce complex signatures that always differed between unrelated individuals and yet were highly reproducible. In contrast, all mother-child pairs tested were identical, as expected from the matrilineal inheritance of mtDNA. Thus, the use of LSSP-PCR to produce D-loop signatures constitutes a powerful new technique for mtDNA-based comparative identity testing.
We report here a polymerase chain reaction (PCR)-based DNA profiling technique that permits Trypanosoma cruzi strain characterization by direct study of infected tissues. This is based on application of a recently developed method of DNA fragment identification, called low-stringency single specific primer PCR (LSSP-PCR), to the study of the variable region of kinetoplast DNA (kDNA) minicircles from T. cruzi Thus, we can translate the intraspecific polymorphism in the nucleotide sequence of kDNA minicircles into a specific and highly reproducible kDNA signature. Comparison with the phenogram obtained by DNA fingerprinting analysis of a set of T. cruzi strains showed good qualitative correlation between the degree of divergence of the LSSP-PCR profiles and the genetic distance between the strains. kDNA signatures of heart tissue from acutely or chronically infected animals revealed perfect concordance with the patterns obtained from cultured parasites for the CL and Colombiana strains but not for the Y strain, which is known to be multiclonal. However, the match was perfect for studies with two clones of the Y strain. We take this as evidence that in some multiclonal strains there is heterogeneity among the clones in the degree of tropism for the heart tissue. Finally, we showed that it is possible to obtain a T. cruzi kDNA signature from the heart of a human patient with chronic Chagasic myocardiopathy. kDNA signatures obtained by LSSP-PCR of sequences amplified from infected tissues constitute a new tool to study the molecular epidemiology of Chagas' disease.
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It has been shown that the mtDNA of the parasitic trematode Schistosoma mansoni is hypervariable in size. We report here that this length variation is due to a large polymorphic minisatellite composed of two types of repeated sequences of 558 bp and 62 bp. Each minisatellite repeat is made up of a large 558-bp component and a variable tandem array of the small 62-bp unit. Of more fundamental interest was the finding that both the 558-bp and 62-bp components have significant homology with a gene, SM750, previously identified in the nuclear genome of S. mansoni. The small 62-bp unit is identical to the nuclear polymorphic repeat element, which is apparently spread throughout the nuclear genome and is abundant among transcripts, in addition to being present in five tandem copies in SM750. The presence, in the S. mansoni mtDNA, of fragments of genes that are present in and transcribed from the nuclear genome raises the question of the origin of these sequences. The arrangement and the variability that the mtDNA minisatellite embodies were explored as an identity test for S. mansoni based on the use of PCR for tallying the relative abundance of the several repeat numbers of the tandem arrays of the 62-bp unit within the minisatellite structure.
A directional size-selected cDNA library constructed from Schistosoma mansoni (Sm) adult worm RNA was used for the generation of expressed sequence tags (EST). From one or both ends of 429 distinct cDNA clones 607 EST were obtained. Of these, only 16% were previously known Sm genes. More than 22% of the clones had matches with entries for other organisms in the databases. These new Sm genes constituted a broad range of transcripts distributed among cytoplasmic structural and regulatory proteins, enzymes, membrane, nuclear and secretory proteins, and proteins with other functions. Almost 33% of the clones had no significant database matches and thus potentially represent Sm-specific genes. Among the latter, several clones, as judged by their redundancy in the library, appear to represent abundant transcripts. The data, taken as a whole, more than double the number of Sm genes identified by nucleotide sequencing and indicate the potential value of the adoption of genome sequencing strategies for the rapid increase in knowledge of complex disease-causing organisms.
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Schistosoma mansoni has 7 pairs of autosomal chromosomes and one pair of sex chromosomes (ZZ for a male worm and ZW for a female), of a haploid genome size of 2.7 x 10(8) bp. We initiated the molecular genetic approach for the detailed characterization and understanding of the evolutionary biology of schistosomes. We have constructed a yeast artificial chromosome (YAC)-library with partially digested parasite genomic DNA, and the chromosome location of each insert was detected by fluorescent in situ hybridization (FISH). The library contains > 2283 clones with an average insert size of 358 kb, which represents a 2.6-fold coverage of the genome (> 7.2 x 10(8) bp). 100 randomly selected YAC clones were localized by FISH and found to be distributed widely among all chromosomes. The assembly of 14 YACs distributed almost the whole region of chromosome 3. Generated expressed sequenced tags (ESTs) derived from a unidirectional cDNA library were also used for further characterization of the YAC inserts. These results indicate that an extensive contig assembly of the entire chromosomes and a reasonably detailed gene map should be feasible in the near future.
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A 285-bp DNA fragment was amplified using the polymerase chain reaction from 38 Leptospira serovars of six different genomic species. The fragments amplified exhibited differential mobilities on nondenaturing polyacrylamide gels resulting from sequence-dependent conformational alterations. Leptospira interrogans serovars could be distinguished from those of other species on this basis.
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A new PCR-based technique, low stringency single specific primer-PCR (LSSP-PCR), has been adapted to produce gene signatures capable of distinguishing human papillomaviruses (HPVs) in clinical specimens. This approach potentially offers a sensitive, inexpensive and high-throughput methodology for precise clinical diagnosis of HPV infections.
Twenty-four fungal feeding-line infections occurred in 17 patients during 1984-1992. Thirteen were receiving long-term home parenteral feeding and, in them, the first infection occurred after a median of 30 months (range 1-120) continuous feeding with a line that had been in situ for a median of 20 months (range 1-37). Four were receiving short-term feeding through a line that had been inserted 1-2 months previously. At the time of the first infection all patients were febrile and most were anaemic (15/16), however a leucocytosis was rare (three of 16). The fungi isolated were Candida albicans(6), Candida parapsilosis(5), Candida glabrata(2), Candida guillermondii(2) and other species (2). In 16 patients, the feeding-line was removed at the time of the first infection and no other treatment was given, and no other complications occurred in eight (50%) of these. In 11, the line was reinserted a median of 7 days after removal (range 1-11). Four patients (24%) developed a Candida infection of the eye 1-8 weeks after the diagnosis, uveitis (2) and endophthalmitis (2) which, in one patient, led to complete blindness in one eye. Two patients had recurrent infections which began within a month of dental therapy. In one, the infections stopped after dental extractions and, in the other, after a dental clearance. An ophthalmoscopic examination should be performed in all patients with a fungal feeding-line infection. Recurrent candidal infections may have a dental origin.
Low-stringency single specific primer PCR (LSSP-PCR) is an extremely simple PCR-based technique that detects single or multiple mutations in gene-sized DNA fragments. A purified DNA fragment is subjected to PCR using high concentrations of a single specific oligonucleotide primer, large amounts of Taq polymerase, and a very low annealing temperature. Under these conditions the primer hybridizes specifically to its complementary region and nonspecifically to multiple sites within the fragment, in a sequence-dependent manner, producing a heterogeneous set of reaction products resolvable by electrophoresis. The complex banding pattern obtained is significantly altered by even a single-base change and thus constitutes a unique "gene signature." Therefore LSSP-PCR will have almost unlimited application in all fields of genetics and molecular medicine where rapid and sensitive detection of mutations and sequence variations is important. The usefulness of LSSP-PCR is illustrated by applications in the study of mutants of smooth muscle myosin light chain, analysis of a family with X-linked nephrogenic diabetes insipidus, and identity testing using human mitochondrial DNA.