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Biomedical subjects

A J Simpson

Publications and source records attributed to A J Simpson.

At least 127 records · Page 7Linked to original sources
PubMed1994

Biomphalaria glabrata: extensive genetic variation in Brazilian isolates revealed by random amplified polymorphic DNA analysis.

To examine the extent of genetic variation in Biomphalaria glabrata, the intermediate host of Schistosoma mansoni, specimens from seven Brazilian isolates were studied. Regions of genomic DNA were amplified using short, arbitrarily selected oligonucleotide primers under low-stringency conditions (random amplified polymorphic DNAs--RAPDs) and analyzed by polyacrylamide gel electrophoresis followed by silver staining. The RAPD profiles of snails of the same isolate were relatively homogeneous, with the majority of bands being common to all individuals studied. In contrast, the profiles of snails from different isolates were quite distinct, with less than 10% of amplified DNA fragments being common to all of the specimens studied. This was found to be true irrespective of the primer, indicating that B. glabrata is remarkably genetically heterogeneous. The data are consistent with the view that genetic drift plays a major role in the genetic structure of populations of B. glabrata due to their ability to reconstitute populations from very small numbers of individuals by self-fertilization. The great variety of polymorphic genetic markers identified in this restricted survey indicates that RAPD analysis may make a major contribution to the study of the genetics of Biomphalaria.

Animals↗
PubMed1994

Partial characterization and kinetics of expression of Sm15, a Schistosoma mansoni tegumental antigen.

Differential antibody screening of an adult Schistosoma mansoni cDNA expression library constructed in lambda gt11 identified a partial cDNA clone, A70. This cDNA encodes a fusion protein recognized by antibodies raised against highly irradiated schistosomula and adult worm tegumental membranes but not by anti-egg antibodies. Anti-tegumental membrane antisera affinity-purified on the A70 cDNA fusion protein were used for Western blotting analysis and indirect immunofluorescence, resulting in the identification of a 15-kDa protein (Sm15) in the tegument of adult worms. This is one of the principal tegumental antigens recognized by antibodies from mice protectively vaccinated with adult worm tegumental membranes. Sm15 is much smaller than the protein encoded by its gene, suggesting that it results from a highly processed precursor. It was found that Sm15 behaves as an integral membrane protein upon partitioning in Triton X-114 and that it is present in worms of 2 weeks or older but not in schistosomula or miracidia. The affinity-purified antibodies also revealed the presence of a 23-kDa antigen in whole-worm homogenates that is apparently coexpressed with Sm15. The 23-kDa antigen was not found associated with membranes and is probably a soluble protein. A further series of Western blots were undertaken using antibodies affinity-purified from serum raised against schistosomula. In this case, the 23- and 15-kDa products were not recognized, but rather soluble proteins ranging from 45- to 150-kDa were detected in almost all larval stages investigated. The results suggest that the precursor is differentially processed during maturation.

Animals↗
PubMed1994

A phylogenetic analysis of Schistosoma haematobium group species based on randomly amplified polymorphic DNA.

Randomly amplified polymorphic DNA (RAPD) profiles were produced using four oligonucleotide primers with genomic DNA from 15 isolates of schistosome. Both inter- and intraspecific variation were noted. Intraspecific variation was greater for two species of the S. haematobium group (S. haematobium and S. intercalatum) than for S. mansoni. The inferred phylogeny placed S. curassoni and S. bovis as sister groups to S. mansoni-S. rodhaini group. S. mattheei and S. leiperi formed a separate lineage. The results confirm that RAPD profiles may be used for both strain and species differentiation and for the generation of phylogenetic trees.

Animals↗
PubMed1994

Low-stringency PCR with diagnostically useful primers for identification of Leptospira serovars.

Primers proposed for the diagnosis of the pathogenic spirochete Leptospira spp. (C. Gravekamp, H. V. D. Kemp, M. Franzen, D. Carrington, G.J. Schoone, G.J.J.M. Van Eys, C. O. R. Everard, R.A. Hartskeel, and W.J. Terpstra, J. Gen. Microbiol. 139:1691-1700, 1993) have been found to produce complex serovar-specific patterns under low-stringency PCR conditions. Such patterns obtained by low-stringency PCR, which maintain the specific band as an internal control, offer, an approach to the standardized identification of Leptospira serovars in clinical laboratories.

Bacteriological Techniques↗
PubMed1994

Assessment of two methods for rapid intrapartum detection of vaginal group B streptococcal colonisation.

AIMS: To compare two methods for the rapid detection of intrapartum vaginal carriage of group B streptococci (Streptococcus agalactiae) with standard culture techniques and to establish their suitability for routine use. METHODS: Vaginal swabs from 266 patients in labour were incubated in glucose broth in an anaerobic atmosphere for four to six hours. The Wellcogen Strep B latex particle agglutination test kit was subsequently used for antigen detection. In the second part of the study swabs from 117 women were assessed for the presence of group B streptococci using the ICON STREP B immuno-concentration assay (Hybritech). Both methods were compared with standard semiquantitative culture on Columbia horse blood agar and Islam's medium. RESULTS: In the first study vaginal carriage of group B streptococci was shown in 38 of 266 (14.3%) patients by culture. Latex particle agglutination with the Wellcogen kit detected 30 of these positive results (sensitivity 78.9%, specificity 100%). In those patients with moderate to heavy colonisation (> 10(4) colony forming units per millilitre) antigen was detected in all (26/26) culture positive patients (sensitivity 100%, specificity 100%). In the second study 16 (13.7%) patients were culture positive. The ICON test detected 11 positive results (sensitivity 68.8%, specificity 100%) and for heavy colonisation (10(5) cfu/ml) detected nine of nine cases (sensitivity 100%, specificity 100%). The ICON test took 10 to 15 minutes to perform. CONCLUSION: These tests are potentially useful for the rapid detection of group B streptococci vaginal colonisation in labour, particularly heavy colonisation. Both tests are insufficiently sensitive to replace standard culture methods.

Agglutination Tests↗
PubMed1994

Rapid silver staining and recovery of PCR products separated on polyacrylamide gels.

A rapid silver-staining procedure for DNA fragments in polyacrylamide gels is described. The time required for band detection is 15 min and the limit of sensitivity 3 pg/mm2. PCR products subjected to this rapid staining protocol are readily recovered from the gel by excision and elution by incubation at 95 degrees C for 20 min. Bands of up to 3 kb have been recovered and reamplified from either recently prepared or dried gels. The rapid staining protocol significantly decreases the processing time required for silver-stained polyacrylamide gels, which is of particular importance in diagnostic situations. The recovery protocol allows individual bands from complex mixtures to be easily recovered for sequencing or probe preparation.

Animals↗
PubMed1993

The use of RAPDs for the analysis of parasites.

There is a lack of sequence information concerning polymorphic loci in parasite genomes. Thus, the use of arbitrary PCR primers under low temperature annealing conditions to generate random amplified polymorphic DNAs (RAPDs) represents an important approach to the study of the structure of parasite populations, their genetic variation as well as improved diagnosis of the diseases they cause. Following the examination of all variables and their effect on the reproducibility of the reaction, we have established a protocol for the analysis of RAPDs that involves amplification at two separate DNA concentrations followed by polyacrylamide gel electrophoresis and silver staining. We find the technique to be sensitive, reproducible, simple and relatively cheap. It has already provided insight into the genetic variation in populations of schistosomes and trypanosomes and is being used to study various other endemic infections. We also use specific primers under low stringency conditions in situations where the objective of the amplification is the detection of a particular sequence and where normal high stringency conditions give a positive/negative answer such as sex determination or diagnosis of blood born infections. Under low stringency conditions, specific amplification products persist but products of low stringency priming are also apparent and serve as a perfect internal control for negative samples.

Animals↗
PubMed1993

The use of RAPDs for the study of the genetic diversity of Schistosoma mansoni and Trypanosoma cruzi.

Arbitrary primers have been used for the production of complex, PCR generated DNA profiles in order to undertake a preliminary random amplified polymorphic DNA (RAPD) analysis of strains (and related species) of two parasitic organisms that are responsible for important diseases endemic in Brazil: Schistosoma mansoni that causes schistosomiasis, and Trypanosoma cruzi that causes Chagas' disease. A relatively low level of polymorphism was found in S. mansoni when strains isolated from different regions of Brazil were compared, with less than 10% of bands exhibiting polymorphism. Comparison of different schistosome species, on the other hand, showed them to be distantly related with very few bands shared by even the more closely related species. Trypanosome strains were found to be much more variable. When strains were compared between zymodemes (groups of parasite strains with the same isoenzyme profiles), a maximum of 7% of bands were found to be common whereas among strains in the same zymodeme a clear characteristic pattern was observed. In the zymodeme most thoroughly studied, it was found that 59% of bands were shared. Band sharing analysis showed that the relationships of strains within a zymodeme correlate with their geographical origin and that the relationship between zymodemes correlates closely with that previously determined by isoenzyme analysis. These preliminary data indicate the ready applicability of RAPD analysis to the study of parasites where largely unexplored genetic variations may have an important bearing on the complexity and diversity of diseases.

Animals↗
PubMed1993

Random amplified polymorphic DNA analysis of Trypanosoma cruzi strains.

DNA extracted from 32 isolates of Trypanosoma cruzi was subjected to polymerase chain reaction amplification using 4 arbitrary primers resulting in relatively complex DNA profiles that include polymorphic markers known as random amplified polymorphic DNAs (RAPDs). The RAPD profiles of 18 strains belonging to zymodeme 1 (Z1) collected from various regions of South America exhibited a consistant pattern and 59 (59%) of the bands produced were present in all Z1 strains. A similar level of consistency was seen in the number of bands shared between 5 Z2 strains, 4 ZB strains and 2 ZC strains. A phenetic analysis of the 5 most different Z1 strains based on band sharing showed that their interrelationships mirrored their geographical origin. Comparison of the RAPD profiles of strains from different zymodemes showed that less than 7% of bands of strains in one zymodeme are present in strains of another zymodeme. Analysis of band sharing using bands present in all strains of a given zymodeme showed ZB and ZC to be closely related and Z1 and Z2 to form distinct groups.

Animals↗
PubMed1993

Structure of the gene encoding a putative Schistosoma mansoni tegumental antigen precursor.

In order to obtain the complete gene encoding the putative precursor of a 15-kDa Schistosoma mansoni tegumental antigen (Sm15), two cDNAs (A70 and A184) and two fragments of independent genomic clones were subcloned and sequenced. The collated sequence contains 4700 nucleotides and represents the full length open reading frame of the gene, encoding a protein of 1032 amino acids with a calculated molecular mass of 116,900. Thus, the gene encodes a much longer protein than that identified in the tegumental membranes, suggesting that it encodes a precursor that is subsequently highly processed. A 964-bp region composed of 5 closely related repeats was found to be present within the translated frame. The predicted protein is highly acidic and there is no indication of hydrophobic domains that may represent transmembrane regions or indicate attachment of a GPI anchor. The coding region has no homologies in the currently available data bases. In the 5' non-transcribed area a copy of the SM alpha repeat family is present. The coding region is preceded by putative CCAAT and TATA boxes that may be involved in the control of expression.

Amino Acid Sequence↗
PubMed1993

The random amplification of polymorphic DNA allows the identification of strains and species of schistosome.

The use of arbitrarily selected primers (10-24 nucleotides) and very low stringency annealing conditions (30 degrees C followed by 40 degrees C) for the polymerase chain reaction amplification of 1.0 ng of schistosome DNA resulted in relatively complex patterns of products. Amongst the primers tested some, for example 5'-TCGTAGCCAA, produced patterns that included bands that were polymorphic between strains of Schistosoma mansoni. Other primers, for example 5'-TCACGATGCA, produced apparently identical products using DNA from 5 S. mansoni strains but highly variable patterns when DNA from different schistosome species was used. The results indicate that the random amplification of polymorphic DNA (RAPD) may be an extremely useful approach to the identification of schistosome strains and species.

Animals↗
PubMed1993

Opening the can of worms: molecular analysis of schistosome populations.

The Schistosomatidae are dioecious digenean parasites of the blood vascular system of vertebrates. Of the 13 genera within the family, only Sehistosoma is associated with humans and, of the mammalian blood flukes, this genus has achieved the greatest geographical distribution and diversification in terms of recognized species and definitive hosts parasitized. In this review, Dave Johnston, Emmanuel Dias Neto, Andy Simpson and David Rollinson consider some recent molecular research that either sheds light on the micro-evolutionary changes occurring within schistosome populations or provides insights into broader, macro-evolutionary questions.

Journal Article↗
PubMed1993

The application of a computerised diagnostic index for ophthalmic practice.

All 2792 patients seen in my private practice between July 1989 and September 1992 and receiving a diagnosis were coded according to the International Classification of Diseases-9 (Clinical Modification), 1979. Any procedures performed were coded according to the American Medical Association Current Procedural Terminology. Patient details and codes were filed by computer using a customised database in Foxpro 2.0 software. Those with minor refractive error or presbyopia alone and those who had no abnormality were excluded from the database. The set-up and maintenance of this diagnostic index is described and the database structure is specified. A modification of the glaucoma section of ICD-9, which better suited my needs, is presented. Examples of the potential information available from such a database is described, including some analysis of this general ophthalmic practice.

Adolescent↗
PubMed1993

Detection of the C protein gene among group B streptococci using PCR.

AIM: To develop a polymerase chain reaction (PCR) for the specific detection of the C protein gene in strains of group B Streptococcus. METHODS: A single primer pair derived from the nucleotide sequence of the IgA binding beta antigen of the C protein complex permitted the specific amplification of a 592 base pair DNA fragment from the C protein gene. After 35 cycles of amplification this product could be detected by agarose gel electrophoresis. Southern blot hybridisation confirmed that this product was the C protein gene. RESULTS: PCR detected the C protein gene in 75 (63%) of 119 strains of group B streptococci analysed. The product was not detected in other Gram positive organisms, showing that this PCR assay was highly specific. The sensitivity of the assay was satisfactory to a dilution of 1 in 10,000 of extracted DNA. CONCLUSIONS: The C protein of group B streptococci is associated with neonatal sepsis. The specific detection of the C protein gene by PCR may help identify which strains are likely to be associated with infection by the organism.

Bacterial Proteins↗