Biomedical subjects

R M Anthony

Publications and source records attributed to R M Anthony.

At least 19 recordsLinked to original sources

PubMed2006

Light emitting diodes for auramine O fluorescence microscopic screening of Mycobacterium tuberculosis.

We describe the simple adaptation of a standard fluorescent microscope for illumination using a 'Royal Blue' Luxeon light emitting diode (LED) and demonstrate that this form of illumination is suitable for the detection of auramine O stained Mycobacterium spp. The low cost, low power consumption, safety and reliability of LEDs makes them attractive alternatives to mercury vapour lamps.

Benzophenoneidum↗

PubMed2005

The use of macroarrays for the identification of MDR Mycobacterium tuberculosis.

The emergence of Mycobacterium tuberculosis (Mtb), resistant to both isoniazid (INH) and rifampicin (RIF) (MDR-TB), is an increasing threat to tuberculosis control programs. Susceptibility testing of Mtb complex isolates by phenotypic methods requires a minimum of 14 days from a primary specimen. This can be reduced significantly if molecular analysis is used. Low density oligonucleotide arrays (macroarrays) have been used successfully for the detection of RIF resistance in Mtb. We describe the use of macroarray technology to identify Mtb complex isolates resistant to INH and/or RIF. The macroarray MDR-Mtb screen has been designed to detect mutations in the RIF resistance determining region (RRDR) of Mtb rpoB and loci in katG and mabA-inhA associated with INH resistance. A panel of Mtb isolates containing 38 different RRDR genotypes, 4 different genotypes within codon 315 of katG and 2 genotypes at mabA-inhA was used to validate the macroarray. The wild type (WT) genotype was correctly identified at all three loci. Of the 37 mutant rpoB genotypes, 36 were correctly detected; the single mutant not detected contained a 9 base insertion. All mutations within katG and mabA-inhA were correctly identified. We conclude that this low cost, rapid system can usefully detect the mutations associated with the vast majority of MDR-Mtb.

Antitubercular Agents↗

PubMed2005

Direct detection of Staphylococcus aureus mRNA using a flow through microarray.

The direct detection of mRNAs from bacterial cultures on a DNA array without amplification and labelling would greatly extend the range of applications suitable for microarray analysis. Here we describe the direct detection of 23S rRNA and seven mRNA species from total Staphylococcus aureus RNA prepared using commercially available RNA purification columns followed by fluorescent detection on a flow through microarray. RNA hybridisation was detected using paired secondary labelled probes directly 5' and 3' to immobilised 60 mers. In this way, we were able to detect the effect of 30-min exposure to antimicrobials on mRNA levels within 3 h after column purification of total RNA without the need for enzymatic manipulation. Specifically the expression of mecA was confirmed in a highly resistant strain and induction of katA and ile-tRNA synthetase genes after exposure to mupirocin could be detected.

Humans↗

PubMed2003

Effect of secondary structure on single nucleotide polymorphism detection with a porous microarray matrix; implications for probe selection.

Oligonucleotide arrays capable of detecting single nucleotide polymorphisms (SNPs) from amplified nucleic acid have many applications. The expected SNP is usually placed approximately in the center of the probe to ensure the maximum shift in Tm between complementary and SNP sequences. Unfortunately, different short probes (< 30 bases) selected using widely accepted criteria do not perform consistently in this type of assay. Here we present a systematic study on the effect of secondary structure on the ability of oligonucleotide probes to detect an SNP, using real-time array monitoring of a porous microarray substrate that incorporates a novel intra-array mixing system. These results demonstrate that, although positioning of an SNP in the middle of the probe is highly destabilizing, the effect of stable secondary structure on the signal obtained is so dramatic that such probes may be very insensitive. Therefore, if the SNP flanking sequence contains significant secondary structure, then more sensitive probes with good specificity may be obtained by positioning the mutation towards one end of the probe.

Artifacts↗

PubMed2003

A methicillin-resistant Staphylococcus aureus outbreak in a new University hospital due to a strain transferred with an infected patient from another city six months previously.

Kocaeli University Medical School was established in 1995. The first methicillin resistant Staphylococcus aureus isolate was detected two years later in a patient transferred from a different city. Six months after this, we detected a small MRSA outbreak in the intensive care unit involving four patients, two of whom had bacteremia, and a staff nasal carrier. All isolates, including the first, appeared to be a single outbreak strain, demonstrated by pulsed field gel electrophoresis profiles which different by at most two bands, identical randomly amplified polymorphic DNA profiles, and identical coagulase gene types by PCR. Antibiogram were identical except that one isolate was additionally resistant to cotrimoxazole. These results show that MRSA isolates can spread between hospitals with infected or colonized patients and can apparently persist in the hospital for six months without causing infection. Screening of asymptomatic patients on wards affected by MRSA or transferred from other hospitals may be helpful in controlling these infections.

Aged↗

PubMed2002

Apparent cross-infection with a single strain of Malassezia pachydermatis on a pig farm.

Twenty-nine isolates of Malassezia pachydermatis were recovered from a single farm of 100 pigs in Croatia. In contrast, 290 farm pigs from other locations (northern parts of Croatia and Slovenia) yielded only two non-lipid dependent isolates of M. pachydermatis using the same swabbing procedure. Ten of the 29 isolates from a single farm had their identity confirmed by karyotyping, and were typed by restriction fragment length polymorphism (RFLP) analysis. All but one of these isolates sub-typed were indistinguishable, one isolate produced a slightly different RFLP profile. Control isolates recovered from dog skin gave RFLP profiles that were easily distinguished from those produced by the pig isolates. These results suggest that a single strain of M. pachydermatis had colonised this pig herd.

Animals↗

PubMed2001

Regional distribution of methamphetamine in autopsied brain of chronic human methamphetamine users.

We measured levels of methamphetamine and those of its metabolite amphetamine in 15 autopsied brain regions of 14 human methamphetamine users. Only slight regional differences were observed in drug concentrations among the brain areas. Although, some redistribution of the drugs probably occurred postmortem, these data suggest that methamphetamine might not be preferentially retained in dopamine-rich brain areas but is heterogenously distributed in brain of chronic human users of the drug. The possible pharmacological actions of methamphetamine in both dopamine-rich and poor brain areas of chronic drug users need to be considered.

Adult↗

PubMed2001

Determining liver stage parasite burden by real time quantitative PCR as a method for evaluating pre-erythrocytic malaria vaccine efficacy.

The detection and quantitation of blood stage parasitaemia is typically used as a surrogate endpoint for estimating the efficacy of vaccines targeted against the hepatic stage, as well as the erythrocytic stage, of the parasite. However, this does not provide an adequate means of evaluating the efficacy of vaccines, which may be only partially effective at the liver-stage. This is a particular concern for effective evaluation of immune enhancement strategies for candidate pre-erythrocytic stage vaccines. Here, we have developed and validated a method for detecting and quantitating liver stage parasites, using the TaqMan fluorescent real-time quantitative PCR system (PE Applied Biosystems). This method uses TaqMan primers designed to the Plasmodium yoelii 18S rRNA gene and rodent GAPDH to amplify products from infected mouse liver cDNA. The technique is highly reproducible as demonstrated with plasmid controls and capable of efficiently quantitating liver-stage parasite burden following a range of sporozoite challenge doses in strains of mice, which differ in their susceptibility to sporozoite infection. We have further demonstrated the capacity of this technique to evaluate the efficacy of a range of pre-erythrocytic stage vaccines. Our data establish this quantitative real-time PCR assay to be a fast and reproducible way of accurately assessing liver stage parasite burden and vaccine efficacy in rodent malaria models.

Animals↗

PubMed2001

DNA array technology and diagnostic microbiology.

Near instantaneous detection of pathogens from clinical material, combined with simultaneous prediction of their antimicrobial resistance profiles, would revolutionize the impact of microbiology on the management of infection. Array-based assays allow a range of characteristics to be rapidly and simultaneously determined. At present these systems have found their primary role as research tools for the monitoring of mRNA expression in the form of DNA microarrays or 'chips'. As fabrication costs reduce and validated targeted arrays are developed, it is inevitable they will be used for more routine applications. Microfluidics offers the exciting possibility of combining purification, amplification and detection in a single disposable device; microarrays are particularly suitable for use within these systems. Arrays will become an important tool for clinical diagnostics.

Animals↗

PubMed2000

The addition of low numbers of 3' thymine bases can be used to improve the hybridization signal of oligonucleotides for use within arrays on nylon supports.

Oligonucleotide arrays can be used for the analysis of microbial nucleic acid. The addition of high numbers of dTTP to the 3' ends of oligonucleotides using terminal transferase has been shown to facilitate membrane binding. This paper demonstrates low numbers of thymine bases added to the 3' end of oligonucleotides during synthesis can improve hybridisation signal intensity where the signal seen with the unmodified oligonucleotides is poor. Thus, the addition of variable numbers of thymine bases to different oligonucleotides allows the production of oligonucleotide arrays producing strong interpretable hybridisation signals.

Antibiotics, Antitubercular↗

PubMed2000

Rapid diagnosis of bacteremia by universal amplification of 23S ribosomal DNA followed by hybridization to an oligonucleotide array.

The rapid identification of bacteria in blood cultures and other clinical specimens is important for patient management and antimicrobial therapy. We describe a rapid (<4 h) detection and identification system that uses universal PCR primers to amplify a variable region of bacterial 23S ribosomal DNA, followed by reverse hybridization of the products to a panel of oligonucleotides. This procedure was successful in discriminating a range of bacteria in pure cultures. When this procedure was applied directly to 158 unselected positive blood culture broths on the day when growth was detected, 125 (79.7%) were correctly identified, including 4 with mixed cultures. Nine (7.2%) yielded bacteria for which no oligonucleotide targets were present in the oligonucleotide panel, and 16 culture-positive broths (10.3%) produced no PCR product. In seven of the remaining eight broths, streptococci were identified but not subsequently grown, and one isolate of Staphylococcus aureus was misidentified as a coagulase-negative staphylococcus. The accuracy, range, and discriminatory power of the assay can be continually extended by adding further oligonucleotides to the panel without significantly increasing complexity or cost.

Bacteremia↗

PubMed1999

Use of the polymerase chain reaction for rapid detection of high-level mupirocin resistance in staphylococci.

The minimum inhibitory concentrations (MICs) of mupirocin were determined by the E test (AB Biodisk, Sweden) and the agar dilution method for 107 staphylococci. The organisms consisted of 34 coagulase-negative staphylococci and 73 methicillin-resistant Staphylococcus aureus. Polymerase chain reaction (PCR) primers designed to amplify a 456 bp region of the plasmid-borne isoleucyl tRNA synthetase gene (ileS-2), responsible for high-level mupirocin resistance in staphylococci, were used on DNA preparations from these isolates. Isolates with high-level mupirocin resistance due to the ileS-2 gene should be PCR positive. There was close correlation between the E test and agar dilution MIC values, with only two strains differing by more than two serial dilutions. Most (51 of 54 strains) of the high-level resistant strains (MIC>256 microg/ml) were resistant to the highest concentration of mupirocin tested (1024 microg/ml). PCR correctly classified all but four (96%) of the isolates in accordance with the results of agar dilution. All four isolates that gave discrepant results were methicillin-resistant Staphylococcus aureus. Two of these were PCR positive, yet the MIC of mupirocin for these strains was <0.06 microg/ml; on prolonged incubation they produced halos within the inhibition zone on agar diffusion testing, suggesting that the phenotypic results may have been erroneous. One of 54 isolates for which the MIC exceeded 256 microg/ml was PCR negative when tested by the original methodology, but a 456 bp product was produced when retested using a lowered annealing temperature. One isolate for which the MIC of mupirocin was 16 microg/ml by agar dilution and 8 microg/ml by the E test was positive by PCR. PCR of the ileS-2 gene is a useful, rapid method for detecting high-level mupirocin resistance in staphylococci.

DNA, Bacterial↗

PubMed1999

Suicide by pipe bomb: a case report.

While lying down, a 23-year-old man detonated an improvised explosive device placed behind his head. The posterior neck and shoulders were singed, and much of the brain was avulsed. Death was due to laceration and partial avulsion of the cerebrum, midbrain, and brain stem. The injuries had a directional nature. Facts derived from the scene investigation and gross dissection, including nature, distribution, and extent of the wounds, in conjunction with preceding medical and social history, allowed for a reasonable reconstruction of the circumstances.

Adult↗

PubMed1999

Failure of bacteriophage typing to detect an inter-hospital outbreak of methicillin-resistant Staphylococcus aureus (MRSA) in Zagreb subsequently identified by random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE).

OBJECTIVE: To establish the extent of inter-hospital spread of methicillin-resistant Staphylococcus aureus (MRSA) in Zagreb and to determine the most suitable method for typing local strains. METHODS: We analyzed a collection of 33 MRSA isolates from three Zagreb hospitals together with five unrelated British MRSA isolates by antibiogram typing, bacteriophage typing, randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) after digestion with Smal restriction endonuclease. Bacteriophage typing was done with the international set of S. aureus typing phages. RAPD and PFGE profiles were analyzed visually and by using the 'GelCompar' computer program. RESULTS: Antibiogram typing provided eight profiles. Thirty (91%) of the 33 Croatian strains of MRSA were non-typable by phage typing. Visual analysis of RAPD products identified six, and visual analysis of PFGE fragments nine, distinct profiles. Computer analysis of RAPD data separated British isolates from the Croatian ones, but did not cluster the visually determined RAPD types. PFGE computer analysis separated British isolates and clustered isolates in concordance with visual interpretation. Thirty-one of the 38 isolates (82%) were visually grouped in the same clusters by both molecular methods. The dominant strain was present in each of the three hospitals. CONCLUSIONS: Bacteriophage typing was unhelpful for the analysis of Croatian MRSA, since most strains were untypable with the international set of bacteriophages. RAPD and PFGE were more successful in typing the organisms and showed evidence of inter-hospital spread of one predominant MRSA strain in all three Zagreb hospitals. Thus RAPD and PFGE proved to be a useful aid in elucidating the epidemiology of MRSA infection in Zagreb hospitals and should be established in Croatia for typing MRSA.

Journal Article↗

PubMed1997

Analysis of an outbreak of non-phage-typeable methicillin-resistant Staphylococcus aureus by using a randomly amplified polymorphic DNA assay.

A cluster of methicillin-resistant Staphylococcus aureus (MRSA) infections among patients on an intensive care unit (ICU) was detected by routine infection control surveillance. In the period from 5 January to 22 June 1995, 10 patients on the ICU and a further 6 patients (5 on one ward that had received colonized patients transferred from the ICU) were affected by MRSA strains with the same antibiotic susceptibility patterns. Seven (44%) of these 16 colonized patients developed MRSA bacteremia. MRSA isolates with the same characteristics were also found on the hands of one member of the ICU staff. The isolates were untypeable by phage typing, but 15 of 17 outbreak strains analyzed genetically had identical randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) profiles. A single strain of MRSA that was nontypeable by phage typing and that was isolated on the ICU on 1 January and six nontypeable and epidemiologically unrelated MRSA isolates all had RAPD profiles distinct from that of the outbreak strain. Implementation of strict infection control measures stopped the further spread of MRSA on the ICU, the affected general ward, and seven other wards that received MRSA carriers from the ICU. Although nontypeable by phage typing and not previously recognized as an epidemic strain, this strain of MRSA was readily transmissible and highly virulent. RAPD typing was found to be a simple, rapid, and effective method for the epidemiological investigation of this outbreak, and performance of typing by this method was simpler and less time-consuming than that of typing by PFGE. RAPD typing may have more general application for the study of S. aureus infections in hospitals.

Bacterial Typing Techniques↗

PubMed1996

Striatal dopamine nerve terminal markers in human, chronic methamphetamine users.

Methamphetamine is a drug that is significantly abused worldwide, Although long-lasting depletion of dopamine and other dopamine nerve terminal markers has been reported in striatum of nonhuman primates receiving very high doses of the psychostimulant, no information is available for humans. We found reduced levels of three dopamine nerve terminal markers (dopamine, tyrosine hydroxylase and the dopamine transporter) in post-mortem striatum (nucleus accumbens, caudate, putamen) of chronic methamphetamine users. However, levels of DOPA decarboxylase and the vesicular monoamine transporter, known to be reduced in Parkinson's disease, were normal. This suggests that chronic exposure to methamphetamine does not cause permanent degeneration of striatal dopamine nerve terminals at the doses used by the young subjects in our study. However, the dopamine reduction might explain some of the dysphoric effects of the drug, whereas the decreased dopamine transporter could provide the basis for dose escalation occurring in some methamphetamine users.

Adult↗

PubMed1996

Application of RAPD and restriction enzyme analysis to the study of oral carriage of Candida albicans.

The genetic similarity of nineteen isolates of Candida albicans from four patients were compared by restriction fragment length polymorphism (RFLP) using EcoRI or HinfI, which both detected five types, and by random amplification of polymorphic DNA (RAPD), which detected three types. Phenotypically unusual isolates also produced distinct patterns with both typing systems demonstrating the carriage of two groups of C. albicans as well as the presence of more than one type in some subjects. Methods of DNA preparation were compared for the production of reproducible patterns; including using the supernatant fluid of boiled intact or spheroplasted cells for RAPD, and DNA precipitated from chloroform extracted cell lysate for RFLP and RAPD. Consistent patterns were produced from the DNA precipitate by RAPD and after an additional precipitation by RFLP, thus removing the necessity for lengthy extraction procedures or the use of toxic chemicals for purification.

AIDS-Related Opportunistic Infections↗

PubMed1995

Characterization of markedly lipid-dependent Malassezia pachydermatis isolates from healthy dogs.

When 244 Malassezia colonies which had been isolated from a colony of Beagle dogs using modified Dixon's agar were sub-cultured on Sabouraud's dextrose agar to determine their lipid dependence, 30 showed poor growth resembling M. furfur, whereas the remainder were typical of M. pachydermatis. Eight of the 10 poor growing isolates selected for further study formed colonies typical of M. pachydermatis after five passages on Sabouraud's dextrose agar at 4 d intervals and two continued to show poor growth. Nine isolates had enzyme profiles identical to those of typical M. pachydermatis isolates, and one resembled M. furfur. However, seven of the poor growing isolates which were karyotyped had patterns typical of M. pachydermatis. Poor growing isolates and their non-lipid-dependent 'revertants' had identical restriction fragment length polymorphism patterns and poly(GT) hybridization profiles. These observations show that some M. pachydermatis isolates grow poorly when sub-cultured onto Sabouraud's dextrose agar and may be incorrectly identified as M. furfur if further studies are not performed.

Anal Canal↗