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1.  Book Review 
Medical History  2007;51(1):122-123.
PMCID: PMC1712373
2.  A slippery disease: a microbiologist's view 
BMJ : British Medical Journal  2006;332(7544):789-790.
PMCID: PMC1420718  PMID: 16575087
4.  Improved diagnosis of reactivated toxoplasmosis. 
Molecular Pathology  1998;51(2):105-109.
AIMS: To identify antigens detected by western blotting in primary Toxoplasma gondii infection and determine their role in diagnosis of reactivated toxoplasmosis. METHODS: Twenty three immunocompromised patients were tested by IgG western blotting. Patients were grouped retrospectively. Group 1 comprised 15 human immunodeficiency (HIV)/AIDS patients and included: group 1A (six patients with clinical and/or serological evidence of reactivation), group 1B (five patients with clinical evidence only), and group 1C (four asymptomatic patients). Group 2 comprised eight non-HIV/AIDS immunocompromised patients with clinical and/or serological evidence of reactivation. Immunocompetent patients (n = 23) with primary toxoplasmosis were a control group used to determine the progression of the antigens detected. RESULTS: In primary toxoplasmosis, antibodies against 6, 20, 22, 23, 25, 28, 29, and 36 kDa antigens predominated. Detection of four or more of the 6, 20, 22, 23, 25, and 36 kDa antigens was considered to be western blot positive. In two group 1A patients, western blotting indicated past infection. During reactivation, this reverted to being western blot positive. Three other group 1A patients were western blot positive. In three of five group 1B patients, western blot positive results improved serological diagnosis of reactivated toxoplasmosis (p < 0.05). In two of five group 1B patients and all four group 1C patients, western blot indicated past infection. In group 2, two of eight patients reverted from a pattern of past infection to western blot positive. Five other patients from group 2 were western blot positive. CONCLUSIONS: Detection of some low molecular weight antigens is diagnostic of reactivated toxoplasmosis. These antigens can be detected even with normal dye test titres and their detection improves the diagnosis of reactivated toxoplasmosis. They might be the result of the release of bradyzoites from ruptured tissue cysts.
PMCID: PMC395619  PMID: 9713595
5.  Do IgA, IgE, and IgG avidity tests have any value in the diagnosis of toxoplasma infection in pregnancy? 
Journal of Clinical Pathology  1998;51(4):312-315.
AIM: To determine the value of tests for specific IgA, IgE, and IgG avidity in diagnosing Toxoplasma gondii infection during pregnancy. METHODS: In a retrospective study, current serological tests (dye test and three IgM assays with different sensitivities) were compared with immunosorbent agglutination assays (ISAGA) for specific IgA and IgE and an IgG avidity enzyme linked immunosorbent assay (ELISA). Patient group 1 comprised six women with definite or probable infection during pregnancy determined by congenital toxoplasmosis or laboratory results. Group 2 comprised seven women infected during or before 11 pregnancies (two consecutive pregnancies in two patients and three in a third). RESULTS: One patient in group 1 seroconverted during pregnancy. IgA ISAGA and avidity confirmed acute infection when confirmatory IgM ELISA remained negative. In five of six patients from group 1, IgA and IgE ISAGA and avidity confirmed acute infection. In group 2, the dye test titre was raised in seven of 11 pregnancies (six of seven patients). Specific IgM and IgA were positive during all 11 pregnancies. IgE ISAGA was positive in only four of 11 pregnancies (three of seven patients), but negative results in the remainder may exclude acute infection. High avidity antibodies indicative of past infection were found in four of 11 pregnancies (two of seven patients). CONCLUSIONS: Each test improved diagnosis or timing of infection but no single test was ideal. The IgA ISAGA was sensitive and detected seroconversion. Positive IgE ISAGA and low avidity both confirmed infection, whereas negative IgE may exclude acute infection. High avidity diagnosed past infection but persistence of low avidity reduced its value to differentiate acute and past infection. Further studies with larger patient groups are needed to determine the optimum diagnostic strategy. These techniques are valuable in complementing existing tests.
PMCID: PMC500678  PMID: 9659246
6.  Specificity and usefulness of an IgE immunosorbent agglutination assay for toxoplasmosis. 
Journal of Clinical Pathology  1995;48(1):64-69.
AIMS--To develop an immunosorbent agglutination assay for the detection of Toxoplasma gondii IgE antibodies (IgE-ISAGA); to assess its specificity; and to determine the role of specific IgE in the diagnosis of current toxoplasma infection. METHODS--Rabbit antihuman IgE capture antibody was adsorbed onto microtitre plates and formaldehyde fixed tachyzoites were used to identify specific antibody. Specificity was assessed in 513 serum samples (blood donor, potentially interfering and difficult, elevated and low total IgE and myeloma). Serum samples (n = 108) from 65 patients with documented toxoplasmosis were tested, as were 26 serum samples from nine pregnant women positive for specific IgM and 30 from 20 HIV positive patients. RESULTS--IgE-ISAGA was highly specific with only three of 513 (0.58%) positive reactions amongst the control groups, one of which (0.19%) was regarded as a false positive. Elevated total IgE did not influence specific IgE results nor did the presence of abnormal immunoglobulin concentrations. Sixty (92.3%) patients with toxoplasma associated lymphadenopathy had specific IgE in one or more samples. Positive or borderline results were obtained in 68 of 77 (88.3%) serum samples taken up to four months after onset and were also detected for up to 11 months in 21 of 31 (67.7%) sera. Of the nine pregnant women with detectable specific IgM, specific IgE was absent in five (12 specimens). Specific IgE was also detected in 10 of 30 (33.3%) serum samples from the 20 HIV positive patients, which was similar to the number with specific IgM. Neither the specific IgE nor IgM tests could distinguish symptomatic from asymptomatic HIV positive patients. CONCLUSIONS--IgE-ISAGA is sensitive, specific, and easy to perform. Although results suggest that specific IgE may be less helpful than previously claimed, specific IgE has a useful role in the diagnosis of current toxoplasma infection when used in conjunction with other tests.
PMCID: PMC502266  PMID: 7706523
7.  Clonal structure of invasive Streptococcus pyogenes in Northern Scotland. 
Epidemiology and Infection  1995;115(2):231-241.
We have used molecular techniques to characterize 51 group A streptococci from Scotland and 17 'serious disease' isolates from other countries, in order to establish the clonal structure of invasive Streptococcus pyogenes strains circulating between 1986 and 1993. Strains were grouped by restriction endonuclease analysis, pulsed field gel electrophoresis and ribotyping patterns, and were examined for the presence of alleles of the speA gene by polymerase chain reaction and DNA sequence analysis. Serious and fatal infections in Scotland were caused by several clones. One clone (9 of 51 strains) was M type 1 and possessed the speA gene allele 2. This was the clone previously identified as causing severe infection in the USA. Another clone (5 of 51 strains) was M type 3 and had speA gene allele 3. In view of the clear association of more than one clone with severe, invasive and fatal infections, horizontal gene exchange between genotypes merits further investigation.
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PMCID: PMC2271403  PMID: 7589263
8.  Allelic variation in the Helicobacter pylori flagellin genes flaA and flaB: its consequences for strain typing schemes and population structure. 
Epidemiology and Infection  1995;114(2):257-266.
Extensive DNA sequence diversity was noted in Helicobacter pylori flagellin genes flaA and flaB. PCR amplified sequences from 49 isolates were digested with AluI, HindIII, MboI or MspI, the resultant patterns were compared between the different isolates and these used to differentiate the isolates from each other. Evidence that the extensive diversity that was found in these genes is the result of reassortment of sequences between strains in the bacterial population is presented, such that a comparatively small number of individual sequence mutations can recombine together in random combinations to form a greater number of distinct alleles. Geographical differences in the predominant patterns in the flaA alleles were also observed and could reflect regional differences either in the human host population or in the bacterial population. In view of the genetic complexity of this species, molecular typing schemes designed to identify related strains may falsely associate strains if the methods do not characterize sufficient genetic sites to exclude chance associations of genetic markers in strains which are actually not closely related to each other.
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PMCID: PMC2271280  PMID: 7705489
9.  Molecular epidemiology of Escherichia coli O157:H7 by pulsed-field gel electrophoresis and comparison with that by bacteriophage typing. 
Journal of Clinical Microbiology  1996;34(4):959-961.
One hundred twenty-four Escherichia coli O157:H7 isolates were characterized by pulse-field gel electrophoresis, bacteriophage typing, and PCR of verotoxin genes. Diversity indices obtained--0.786 for phage types and 0.987 for pulsed-field gel electrophoresis types--demonstrated that phage typing falls below the critical value (0.9) required for confident interpretation of results.
PMCID: PMC228925  PMID: 8815116
10.  Rifampin resistance in Neisseria meningitidis due to alterations in membrane permeability. 
Rifampin-resistant (Rifr) Neisseria meningitidis strains are known to have single point mutations in the central conserved regions of the rpoB gene. We have demonstrated two distinct resistance phenotypes in strains with identical mutations in this region, an intermediate level of resistance in Rifr clinical isolates and a high level of resistance in mutants selected in vitro. The possible role of membrane permeability in the latter was investigated by measuring MICs in the presence of Tween 80; values for high-level-resistance mutants were reduced to intermediate levels, whereas those for intermediate-level-resistance strains were unaffected. The highly resistant mutants were also found to have increased resistance to Triton X-100 and gentian violet. Sequencing of the meningococcal mtrR gene and its promoter region (which determine resistance to hydrophobic agents in Neisseria gonorrhoeae) from susceptible or intermediate strains and highly resistant mutants generated from them showed no mutation within this region. Two-dimensional gel electrophoresis of two parent and Rif mutant strains showed identical shifts in the pI of one protein, indicating that differences between the parent and the highly Rifr mutant are not confined to the rpoB gene. These results indicate that both permeability and rpoB mutations play a role in determining the resistance of N. meningitidis to rifampin.
PMCID: PMC163174  PMID: 8851587
11.  Population genetics of Mycobacterium tuberculosis complex in Scotland analysed by pulsed-field gel electrophoresis. 
Epidemiology and Infection  1995;114(1):153-160.
The results of typing of 121 strains in the Mycobacterium tuberculosis complex by PFGE are presented. Every isolate from patients in Scotland over a 3-month period for M. tuberculosis and for 1 year for M. bovis were included along with several laboratory strains including those of BCG. The PFGE results suggest that the population structure of all the strains in this complex is distinctly simple with limited genetic diversity and also suggest that M. bovis is not a distinct species.
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PMCID: PMC2271342  PMID: 7867734
12.  Incidence and spread of Haemophilus influenzae on an Antarctic base determined using the polymerase chain reaction. 
Epidemiology and Infection  1995;114(1):93-103.
A PCR-based method of detecting Haemophilus influenzae in cultures inoculated from throat swabs was evaluated using samples from groups of laboratory staff and medical students and then applied to samples originating from the closed human community of an Antarctic research station. Suitable PCR primers to an H. influenzae gene (ompP2) were used to amplify the gene from DNA preparations made from mixed growth on chocolate agar with added vancomycin. PCR product was reamplified and subjected to restriction endonuclease digestion to allow temporal and spatial mapping of strains over an 8-month period. Eleven different strains of H. influenzae were detected. One particular strain was detected in a third of the base members.
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PMCID: PMC2271340  PMID: 7867747
13.  Genetic heterogeneity of M type 3 group A streptococci causing severe infections in Tayside, Scotland. 
Journal of Clinical Microbiology  1996;34(1):196-198.
To explain the worldwide increase in the frequency of severe infections by group A streptococci, molecular techniques are increasingly being employed to evaluate the genetic relationships of strains. We used restriction endonuclease analysis, pulsed-field gel electrophoresis (PFGE), the PCR, ribotyping, and DNA sequence analysis in a study of 13 group A streptococci isolated from a cluster of cases of serious infections over a 3-month period in Tayside, Scotland. Eight of the strains were M type 3; molecular characterization identified two subclones. The first, displaying PFGE profile 4, has been observed in Northern Scotland and has been circulating in New Zealand for over a decade. The second subclone has been documented only in the United Kingdom; it was first seen in 1993 in Scotland. Sequence analysis of emm-3 genes further differentiated the PFGE 4 subclone. DNA sequence analysis of virulence factors supports the suggestion that they may have recently been acquired by horizontal gene transfer.
PMCID: PMC228760  PMID: 8748303
14.  The role of a nested polymerase chain reaction in the diagnosis of Pneumocystis carinii pneumonia 
Clinical Molecular Pathology  1995;48(6):M347-M350.
Aim—To compare the techniques and results of a nested PCR and an immunofluorescence assay (IFA) for the detection of Pneumocystis carinii infection; to consider the role of the nested PCR in the diagnosis of P carinii pneumonia (PCP).
Methods—Serial dilutions of two known P carinii positive samples were tested by IFA and PCR to determine their relative sensitivities. Seventy eight respiratory samples (15 from 11 patients with HIV infection/acquired immunodeficiency syndrome (AIDS) and 63 from 42 patients with other forms of immunodeficiency) were tested using both assays, and the costs and technical requirements of each assay were assessed.
Results—The PCR had a greater relative sensitivity over the IFA of 2 × 101 to 2 × 103 fold in a postmortem lung sample and 2 × 105 to 2 × 106 fold in a bronchoalveolar lavage sample from a patient with PCP. P carinii was detected in all 15 samples from the patients with HIV/AIDS by both IFA and PCR. Of the 63 samples from the patients with immunodeficiencies other than HIV/AIDS, the PCR was more sensitive than IFA.
Conclusions—The nested PCR is a more sensitive assay than the IFA. It may be useful in the diagnosis of PCP in patients with immunodeficiencies other than HIV/AIDS. Similarly, PCR may be of benefit for this patient group as less invasive specimens are needed. PCR has an increasing role to play in the diagnosis of PCP in the routine laboratory.
PMCID: PMC408003  PMID: 16696036
Pneumocystis carinii; PCR; immunofluorescence assay; acquired immunodeficiency syndrome
15.  Molecular epidemiology of recent United Kingdom isolates of Neisseria meningitidis serogroup C. 
Epidemiology and Infection  1994;113(1):53-65.
The genomes of 34 recent United Kingdom isolates of Neisseria meningitidis serogroup C were examined by restriction enzyme digestion and by conventional and pulsed-field gel electrophoresis (PFGE). Strains were assigned to groups on the basis of the Dice similarity coefficient; strains with values of > 92% were considered to be clonally related. Twelve clones were identified by PFGE. Strains of two clonal groups predominated. Restriction endonuclease analyses using the 'high frequency cleavage' endonuclease Stu I and conventional electrophoresis gave 11 groups; in general it had lower resolving power than PFGE.
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PMCID: PMC2271220  PMID: 8062880
16.  The diagnosis of Lyme disease. 
PMCID: PMC1295192  PMID: 7636815
19.  An analysis of the diversity of Haemophilus parainfluenzae in the adult human respiratory tract by genomic DNA fingerprinting. 
Epidemiology and Infection  1993;111(1):89-98.
A method for typing Haemophilus species is described, based on the analysis of genomic DNA from Haemophilus parainfluenzae. The DNA was extracted by a rapid method and digested with the restriction enzyme BamHI to provide a characteristic 'fingerprint'. The pattern of fragments in the ranges 1-1.6 kb, 1.6-2 kb and 2-3 kb were used to produce a numerical profile of each isolate. In total 97 isolates were examined; 88 from throat swab material isolated from the 15 members of a British Antarctic Survey base and 9 type strains. Seventy-two of the 88 antarctic isolates were H. parainfluenzae and were found to be very diverse, comprising 41 identifiable strains with up to 5 strains being isolated from a single throat swab sample. There was evidence for both carriage and transmission within the isolated community. The technique provided a highly discriminatory method for characterizing Haemophilus strains which is suitable for epidemiological studies.
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PMCID: PMC2271197  PMID: 8348936
20.  Molecular characterization of rifampin-resistant Neisseria meningitidis. 
Primers were designed to amplify the rpoB gene of Neisseria meningitidis. The region of the gene amplified covered clusters I and II of the rifampin resistance (Rifr) mutation sites identified in Escherichia coli. DNAs from six Rifr isolates and 21 rifampin-susceptible isolates from the United Kingdom representing a number of serogroups were amplified and sequenced. All six Rifr isolates had identical DNA sequences and the same amino acid change, a His to an Asn change at position 35 (H35N). This His residue is equivalent to the His residue at position 526 in E. coli, one of the known Rifr mutation sites. DNAs from an additional six Rifr mutations generated in vitro were amplified and sequenced. Three had H35Y changes, one had an H35R change, one had an H35N change and one had an S40F change. The predominance of mutations at the His residue at position 35 in Rifr N. meningitidis isolates suggests that it plays a critical role in the selection of antibiotic-resistant variants. All six Rifr isolates belonged to the same clonal group when analyzed by restriction enzyme analysis and pulsed-field gel electrophoresis. These data suggest that a single clone of Rifr N. meningitidis is present and widespread throughout the United Kingdom.
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PMCID: PMC188195  PMID: 8092823
21.  Osteotomy as an indicator of antiseptic surgical practice. 
Medical History  1994;38(2):178-188.
PMCID: PMC1036843  PMID: 8007752
23.  Characterization of enterococcal isolates by restriction enzyme analysis of genomic DNA. 
Epidemiology and Infection  1992;109(1):69-80.
A restriction enzyme analysis (REA) of chromosomal DNA for the intra-species characterization of enterococci is reported. The DNA was extracted by a rapid method and digested with the restriction enzyme Sal I to provide a characteristic 'fingerprint' consisting of 10-20 bands in the 1.6-5.0 kb range. One hundred and eighty enterococcal isolates were examined; 5 were type strains, 15 from an out-patient clinic and 160 from a geographically isolated British Antarctic Survey Base. The epidemiologically unrelated out-patient clinic isolates gave readily distinguishable patterns, whereas isolates from the geographically isolated community showed evidence of colonization. This technique provided a highly discriminatory method of isolate characterization for Enterococcus faecalis, E. faecium and E. durans suitable for epidemiological studies. A sample of isolates were probed with 16 + 23 S ribosomal RNA from Escherichia coli. Discrimination between isolates was poorer than with REA, although good correlation was observed between the results of the two techniques.
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PMCID: PMC2272234  PMID: 1379935
25.  The population structure and transmission of Escherichia coli in an isolated human community; studies on an Antarctic base. 
Epidemiology and Infection  1991;107(3):537-542.
The population structure and transmission of Escherichia coli in a small group of individuals isolated for 26 weeks on an Antarctic base were studied by multilocus electrophoresis of eight enzymes and plasmid analysis. Two hundred and sixty-nine strains were isolated. They were grouped into 60 allozyme types (ETs). Half of these ETs were only isolated once; others were repeatedly isolated from single subjects. Eleven were found in more than one subject and the pattern of the occurrence of some of them was considered to provide evidence of their spread from subject to subject.
PMCID: PMC2272106  PMID: 1752303

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