Biomedical subjects

Paul T Monis

Publications and source records attributed to Paul T Monis.

16 recordsLinked to original sources

PubMed2006

Rapid, sensitive, and discriminating identification of Naegleria spp. by real-time PCR and melting-curve analysis.

The free-living amoeboflagellate genus Naegleria includes one pathogenic and two potentially pathogenic species (Naegleria fowleri, Naegleria italica, and Naegleria australiensis) plus numerous benign organisms. Monitoring of bathing water, water supplies, and cooling systems for these pathogens requires a timely and reliable method for identification, but current DNA sequence-based methods identify only N. fowleri or require full sequencing to identify other species in the genus. A novel closed-tube method for distinguishing thermophilic Naegleria species is presented, using a single primer set and the DNA intercalating dye SYTO9 for real-time PCR and melting-curve analysis of the 5.8S ribosomal DNA gene and flanking noncoding spacers (ITS1, ITS2). Collection of DNA melting data at close temperature intervals produces highly informative melting curves with one or more recognizable melting peaks, readily distinguished for seven Naegleria species and the related Willaertia magna. Advantages over other methods used to identify these organisms include its comprehensiveness (encompassing all species tested to date), simplicity (no electrophoresis required to verify the product), and sensitivity (unambiguous identification from DNA equivalent to one cell). This approach should be applicable to a wide range of microorganisms of medical importance.

Animals↗

PubMed2005

Nucleic acid amplification-based techniques for pathogen detection and identification.

Nucleic acid amplification techniques have revolutionised diagnostic and research industries. Current technologies that allow the detection of amplification in real-time are fast becoming industry standards, particularly in a diagnostic context. In this review, we describe and explore the application of numerous real-time detection chemistries and amplification techniques for pathogen detection and identification, including the polymerase chain reaction, nucleic acid sequence-based amplification, strand displacement amplification and the ligase chain reaction. The emergence of newer technologies, such as lab-on-a-chip devices and photo-cleavable linkers, is also discussed.

DNA, Bacterial↗

PubMed2005

Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis.

Following the initial report of the use of SYBR Green I for real-time polymerase chain reaction (PCR) in 1997, little attention has been given to the development of alternative intercalating dyes for this application. This is surprising considering the reported limitations of SYBR Green I, which include limited dye stability, dye-dependent PCR inhibition, and selective detection of amplicons during DNA melting curve analysis of multiplex PCRs. We have tested an alternative to SYBR Green I and report the first detailed evaluation of the intercalating dye SYTO9. Our findings demonstrate that SYTO9 produces highly reproducible DNA melting curves over a broader range of dye concentrations than does SYBR Green I, is far less inhibitory to PCR than SYBR Green I, and does not appear to selectively detect particular amplicons. The low inhibition and high melting curve reproducibility of SYTO9 means that it can be readily incorporated into a conventional PCR at a broad range of concentrations, allowing closed tube analysis by DNA melting curve analysis. These features simplify the use of intercalating dyes in real-time PCR and the improved reproducibility of DNA melting curve analysis will make SYTO9 useful in a diagnostic context.

Base Pairing↗

PubMed2005

Emerging technologies for the detection and genetic characterization of protozoan parasites.

The development and adaptation of new technologies for the genetic characterization and identification of parasites continue to accelerate, providing an increasing number of research and analytical tools. We review emerging technologies that have applications in this area, including real-time PCR and microarrays, and discuss the fundamental principles of some of these technologies and how they are applied to characterize parasites. We give special consideration to the application of genetic data to biological questions, where selection of the most appropriate technique depends on the biological question posed by the investigator.

Animals↗

PubMed2005

Culture-independent techniques for rapid detection of bacteria associated with loss of chloramine residual in a drinking water system.

Chloramination is often the disinfection regimen of choice for extended drinking water systems. However, this process is prone to instability due to the growth of nitrifying bacteria. This is the first study to use alternative approaches for rapid investigation of chloraminated drinking water system instability in which flow cytometric cell sorting of bacteria with intact membranes (membrane-intact fraction) (BacLight kit) or with active esterases (esterase-active fraction) (carboxyfluorescein diacetate) was combined with 16S rRNA gene-directed PCR and denaturing gradient gel electrophoresis (DGGE). No active bacteria were detected when water left the water treatment plant (WTP), but 12 km downstream the chloramine residual had diminished and the level of active bacteria in the bulk water had increased to more than 1 x 10(5) bacteria ml(-1). The bacterial diversity in the system was represented by six major DGGE bands for the membrane-intact fraction and 10 major DGGE bands for the esterase-active fraction. PCR targeting of the 16S rRNA gene of chemolithotrophic ammonia-oxidizing bacteria (AOB) and subsequent DGGE and DNA sequence analysis revealed the presence of an active Nitrosospira-related species and Nitrosomonas cryotolerans in the system, but no AOB were detected in the associated WTP. The abundance of active AOB was then determined by quantitative real-time PCR (qPCR) targeting the amoA gene; 3.43 x 10(3) active AOB ml(-1) were detected in the membrane-intact fraction, and 1.40 x 10(4) active AOB ml(-1) were detected in the esterase-active fraction. These values were several orders of magnitude greater than the 2.5 AOB ml(-1) detected using a routine liquid most-probable-number assay. Culture-independent techniques described here, in combination with existing chemical indicators, should allow the water industry to obtain more comprehensive data with which to make informed decisions regarding remedial action that may be required either prior to or during an instability event.

Bacteria↗

PubMed2005

Legionella confirmation using real-time PCR and SYTO9 is an alternative to current methodology.

The currently accepted culture techniques for the detection of Legionella spp. in water samples (AS/NZS 3896:1998 and ISO 11731 standard methods) are slow and laborious, requiring from 7 to 14 days for a result. We describe a fully validated rapid confirmation technique that uses real-time PCR incorporating the intercalating dye SYTO9 for the direct identification of primary cultures, significantly decreasing turnaround time and allowing faster remedial action to be taken by the industry.

Latex Fixation Tests↗

PubMed2005

Environmental temperature controls Cryptosporidium oocyst metabolic rate and associated retention of infectivity.

Cryptosporidium is a significant cause of water-borne enteric disease throughout the world and represents a challenge to the water industry and a threat to public health. In this study we report the use of a cell culture-TaqMan PCR assay to measure oocyst inactivation rates in reagent-grade and environmental waters over a range of temperatures. While oocysts incubated at 4 degrees C and 15 degrees C remained infective over the 12-week holding period, we observed a 4 log(10) reduction in infectivity for both 20 and 25 degrees C incubation treatments at 12 and 8 weeks, respectively, for all water types examined, a faster rate of inactivation for oocysts than previously reported. This temperature-dependent inactivation was further investigated using a simple and rapid ATP assay described herein. Time course experiments performed in reagent-grade water at incubation temperatures of 4, 15, 20, 25, 30, and 37 degrees C identified a close relationship between oocyst infectivity and oocyst ATP content, demonstrating that temperature inactivation at higher temperatures is a function of increased oocyst metabolic activity. While water quality did not affect oocyst inactivation, biological antagonism appears to be a key factor affecting oocyst removal from environmental waters. Both the cell culture-TaqMan PCR assay and the ATP assay provide a sensitive and quantitative method for the determination of environmental oocyst inactivation, providing an alternative to the more costly and time-consuming mouse infection assay. The findings presented here relating temperature to oocyst inactivation provide valuable information for determining the relative risks associated with Cryptosporidium oocysts in water.

Adenosine Triphosphate↗

PubMed2004

Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP.

A PCR-RFLP genotyping tool was developed and used to characterise morphologically identical isolates of Giardia duodenalis from a variety of host species. Primers were designed to amplify a 432bp region of the glutamate dehydrogenase gene (gdh) from genetic Assemblages AI, AII, BIII, BIV, C, D and E of G. duodenalis. DNA extracted from cultured Giardia trophozoites, Giardia cysts purified from faeces and directly from whole faeces was amplified and sequenced at the gdh and 18SrDNA loci. The gdh sequences were identical with published gdh sequences for each assemblage with a few exceptions. However, in some cases genotyping results obtained using gdh differed from 18SrDNA genotyping results. From gdh sequence information a PCR-RFLP profile was identified for each of the genetic assemblages. PCR-RFLP is a reproducible, reliable and sensitive method for genotyping Giardia. Eight human, 12 cat, 9 dog and 16 cattle faecal isolates were genotyped using PCR-RFLP. This method allows G. duodenalis isolates from human-beings, their companion animals and livestock to be genotyped directly from faeces, leading to valuable information about Giardia genotypes in population without the need for in vitro/in vivo amplification.

Alleles↗

PubMed2004

Cyst morphology and sequence analysis of the small subunit rDNA and ef1 alpha identifies a novel Giardia genotype in a quenda (Isoodon obesulus) from Western Australia.

Sequence analysis of the small subunit ribosomal DNA (SSU-rDNA) and elongation factor 1 alpha (ef1 alpha) was performed on Giardia cysts isolated from faeces collected from a quenda (Isoodon obesulus) in the southwest of Western Australia. The SSU-rDNA and ef1 alpha were sequenced in their entirety and correspondingly aligned with the published sequence information of other known species and genotypes of Giardia. Phylogenetic analysis of the SSU-rDNA and ef1 alpha sequences identified the quenda isolate as a novel genotype of Giardia not previously reported. We believe that this quenda Giardia isolate constitutes a distinct species, which may be endemic within the Australian native fauna.

Animals↗

PubMed2004

Detection and significance of the potentially pathogenic amoeboflagellate Naegleria italica in Australia.

Thermophilic amoeboflagellates in the genus Naegleria include both virulent and benign species. One of the less studied species, N. italica, has not been detected in the environment since the first reports from Italy in the 1980s; its virulence is known only from infection of laboratory mice. Two recent strains from recreational water in Western Australia (AWQC NG960, NG961) were tentatively identified as N. italica from the characteristic mobilities of seven isozymes. Sequences of the 5.8S rRNA gene and its flanking ITS aligned with a 380+bp length of the published sequence for N. italica with 98% identity. Differences from the type strain were confined to ITS2. Shorter alignments (<320 bp) were observed with other Naegleria species, corresponding to conserved regions of the 5.8S gene and ITS. Unlike the European type strain of N. italica, the Australian isolates failed to infect laboratory mice intranasally, confirming that infectivity of this species is variable and often lower than in N. fowleri.

Amebiasis↗

PubMed2003

Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR.

SYBR Green I (SG) is widely used in real-time PCR applications as an intercalating dye and is included in many commercially available kits at undisclosed concentrations. Binding of SG to double-stranded DNA is non-specific and additional testing, such as DNA melting curve analysis, is required to confirm the generation of a specific amplicon. The use of melt curve analysis eliminates the necessity for agarose gel electrophoresis because the melting temperature (T(m)) of the specific amplicon is analogous to the detection of an electrophoretic band. When using SG for real-time PCR multiplex reactions, discrimination of amplicons should be possible, provided the T(m) values are sufficiently different. Real-time multiplex assays for Vibrio cholerae and Legionella pneumophila using commercially available kits and in-house SG mastermixes have highlighted variability in performance characteristics, in particular the detection of only a single product as assessed by T(m) analysis but multiple products as assessed by agarose gel electrophoresis. The detected T(m) corresponds to the amplicon with the higher G+C% and larger size, suggesting preferential binding of SG during PCR and resulting in the failure to detect multiple amplicons in multiplex reactions when the amount of SG present is limiting. This has implications for the design and routine application of diagnostic real-time PCR assays employing SG.

Bacterial Proteins↗

PubMed2003

A comparative study of carboxyfluorescein diacetate and carboxyfluorescein diacetate succinimidyl ester as indicators of bacterial activity.

Staining bacteria with esterified fluorogenic substrates followed by flow cytometric analysis offers a means for rapid detection of metabolically active bacteria. Flow cytometry (FCM) was used to assess carboxyfluorescein diacetate (CFDA) and carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) as indicators of bacterial activity for cultured bacteria, including Aeromonas hydrophila, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis and bacteria from environmental waters. In theory, CFDA/SE should be a better indicator of metabolic bacterial activity compared to CFDA due to greater intracellular retention of the fluorescent product. Qualitative and quantitative analysis of exponential phase cultures, mixtures of active and inactive cells and bacteria from environmental waters revealed CFDA was successful in detecting active bacteria, whereas CFDA/SE was not. CFDA/SE labelled inactive cells with intensities equal to that of the active population and could not even discriminate between bacteria in exponential phase growth and a fixed cell preparation. We propose that the specific mode of action of the succinimidyl ester (SE) group in combination with the nonenzymatic aqueous hydrolysis of the CFDA moiety results in the nonspecific labelling of all cells, irrespective of their metabolic state. This study shows that CFDA/SE is a poor marker of bacterial activity.

Bacteria↗

PubMed2003

Enumeration of water-borne bacteria using viability assays and flow cytometry: a comparison to culture-based techniques.

Maintaining optimal conditions in catchments or distribution systems relies heavily on water authorities having access to rapid and accurate water quality data, including an indication of bacteriological quality. In this study, the BacLight bacterial viability kit and carboxyfluorescein diacetate (CFDA) were coupled with flow cytometry (FCM) for rapid detection of physiologically active bacteria from raw and potable waters taken from various locations around South Australia. Results were compared to the direct viable count (DVC) and quantitative DVC (qDVC), in addition to the culture-based methods of the heterotrophic plate count (HPC) and a commercial SimPlate technique. Raw and potable water analysis revealed that DVC and culture-based techniques reported significantly fewer viable bacteria compared to the number of physiologically active bacteria detected using the rapid FCM assays, where this difference appeared to be nonlinear across different samples. Inconclusive results were obtained using qDVC as a viability assay. In particular, HPC results were 2-4 log orders of magnitude below that reported by the FCM assays for raw waters. Few bacteria in potable waters examined were culturable by HPC, even though FCM assays reported between 5.56 x 10(2) and 3.94 x 10(4) active bacteria ml(-1). These differences may be attributed to the presence of nonheterotrophic bacteria, sublethal injury or the adoption of an active but nonculturable (ABNC) state.

Bacteria↗

PubMed2003

Genetic diversity within the morphological species Giardia intestinalis and its relationship to host origin.

A genetic analysis of Giardia intestinalis, a parasitic protozoan species that is ubiquitous in mammals worldwide, was undertaken using organisms derived from a variety of mammalian hosts in different geographical locations. The test panel of 53 Giardia isolates comprised 48 samples of G. intestinalis, including representatives of all known genetic subgroups, plus an isolate of G. ardeae and four isolates of G. muris. The isolates were compared by allozymic analysis of electrophoretic data obtained for 21 cytosolic enzymes, representing 23 gene loci. Neighbour Joining analysis of the allelic profiles supported the monophyly of G. intestinalis but showed that the species encompasses a rich population substructure. Seven major clusters were evident within G. intestinalis, corresponding to lineages designated previously as genetic assemblages A-G. Some genotypes, e.g. those defining assemblage A, are found in divergent host species and may be zoonotic. However other genotypes, e.g. those defining assemblages C-G, appear to be confined to particular hosts or host groups. The findings reinforce other evidence that G. intestinalis, which was defined on the basis of morphological criteria only, is a species complex.

Alleles↗

PubMed2003

Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection.

Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log(10) units) with LP-UV (20 mJ/cm(2)) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log(10) unit being inactivated. These results demonstrate the inability of MIOX to inactivate Cryptosporidium. The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water.

Animals↗

PubMed2002

Molecular biology techniques in parasite ecology.

Molecular techniques are increasingly being used to study the ecology of a variety of organisms. These techniques represent important tools for the study of the systematics, population genetics, biogeography and ecology of parasites. Here, we review the techniques that have been employed to study the ecology and systematics of parasites (including bacteria and viruses). Particular emphasis is placed on the techniques of isoenzyme electrophoresis, in situ hybridisation and nucleic acid amplification to characterise parasite/microbial communities. The application of these techniques will be exemplified using ticks, bacterial endosymbionts and parasitic protozoa.

Animals↗