ORCID Profile
0000-0002-5869-8283
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James Cook University
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Publisher: Elsevier BV
Date: 11-2007
DOI: 10.1016/J.IJFOODMICRO.2007.08.007
Abstract: Sago starch is an important source of dietary carbohydrates in lowland Papua New Guinea. Over the past 30 years there have been sporadic reports of severe illness following consumption of sago starch. A common assumption is that fungal metabolites might be associated with the illness, leading to the need for a more thorough investigation of the mycoflora of sago starch. Sago starch was collected from areas of high sago consumption in Papua New Guinea for fungal analysis (69 s les). Storage methods and duration were recorded at the time of collection and pH on arrival at the laboratory. Yeasts were isolated from all s les except two, ranging from 1.2 x 10(3) to 8.3 x 10(7) cfu/g. Moulds were isolated from 65 of the 69 s les, ranging from 1.0 x 10(2) to 3.0 x 10(6) cfu/g. Of 44 s les tested for ergosterol content, 42 s les showed the presence of fungal biomass. Statistical analyses indicated that sago starch stored for greater than five weeks yielded significantly higher ergosterol content and higher numbers of moulds than sago stored for less than five weeks. The method of storage was also shown to influence mould numbers with storage in natural woven fibre containers returning significantly greater numbers than present in other storage methods tested. Potentially mycotoxigenic genera of moulds including Aspergillus and Penicillium were commonly isolated from sago starch, and as such storage factors that influence the growth of these and other filamentous fungi might contribute to the safety of traditional sago starch in PNG.
Publisher: MDPI AG
Date: 10-02-2019
DOI: 10.3390/TROPICALMED4010033
Abstract: Papua New Guinea (PNG) has a high burden of tuberculosis (TB), including drug-resistant TB (DR-TB). DR-TB has been identified in patients in Western Province, although there has been limited study outside the provincial capital of Daru. This study focuses on the Balimo region of Western Province, aiming to identify the proportion of DR-TB, and characterise Mycobacterium tuberculosis (MTB) drug resistance-associated gene mutations. Sputum s les were investigated for MTB infection using published molecular methods. DNA from MTB-positive s les was lified and sequenced, targeting the rpoB and katG genes to identify mutations associated with rif icin and isoniazid resistance respectively. A total of 240 sputum s les were collected at Balimo District Hospital (BDH). Of these, 86 were classified as positive based on the results of the molecular assays. For s les where rpoB sequencing was successful, 10.0% (5/50, 95% CI 4.4–21.4%) were considered rif icin-resistant through detection of drug resistance-associated mutations. We have identified high rates of presumptive DR-TB in the Balimo region of Western Province, PNG. These results emphasise the importance of further surveillance, and strengthening of diagnostic and treatment services at BDH and throughout Western Province, to facilitate detection and treatment of DR-TB, and limit transmission in this setting.
Publisher: Springer Science and Business Media LLC
Date: 13-11-2019
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C5DT04885K
Abstract: Dinuclear ruthenium( ii ) complexes containing one inert and one labile metal centre have been synthesised and their biological properties examined in bacterial and eukaryotic cells.
Publisher: Wiley
Date: 03-06-2015
Abstract: Ruthenium(II) complexes containing the tetradentate ligand bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane ("bbn " n=10 and 12) have been synthesised and their geometric isomers separated. All [Ru(phen)(bbn )](2+) (phen=1,10-phenanthroline) complexes exhibited excellent activity against Gram-positive bacteria, but only the cis-α-[Ru(phen)(bb12 )](2+) species showed good activity against Gram-negative species. In particular, the cis-α-[Ru(phen)(bb12 )](2+) complex was two to four times more active than the cis-β-[Ru(phen)(bb12 )](2+) complex against the Gram-negative strains. The cis-α- and cis-β-[Ru(phen)(bb12 )](2+) complexes readily accumulated in the bacteria but, significantly, showed the highest level of uptake in Pseudomonas aeruginosa. Furthermore, the accumulation of the cis-α- and cis-β-[Ru(phen)(bb12 )](2+) complexes in P. aeruginosa was considerably greater than in Escherichia coli. The uptake of the cis-α-[Ru(phen)(bb12 )](2+) complex into live P. aeruginosa was confirmed by using fluorescence microscopy. The water/octanol partition coefficients (log P) were determined to gain understanding of the relative cellular uptake. The cis-α- and cis-β-[Ru(phen)(bbn )](2+) complexes exhibited relatively strong binding to DNA (Kb ≈10(6) M(-1) ), but no significant difference between the geometric isomers was observed.
Publisher: Elsevier BV
Date: 10-2004
Publisher: Elsevier BV
Date: 12-2007
DOI: 10.4315/0362-028X-70.12.2868
Abstract: Sago starch is an important food in lowland Papua New Guinea. Extraction of the starch from the palm and storage were performed by way of traditional methods that have been used for thousands of years. Currently, very little is known about the microbiology of sago starch. Sago s les were collected from areas of high starch utilization and analyzed for the presence of bacterial pathogens and indicator organisms. Storage methods and duration were recorded at the time of collection, and pH and water activity on arrival at the laboratory. Sago starch was found to harbor high levels of fecal contamination, as well as various food pathogens including Salmonella, Bacillus cereus, and coagulase-positive staphylococci. Clostridium perfringens was only present infrequently in s les and in very low numbers, while Listeria monocytogenes was not isolated from sago starch. The presence of high levels of fecal contamination in sago starch is of particular concern, and may contribute to diarrheal disease in rural Papua New Guinea.
Publisher: Royal Society of Chemistry (RSC)
Date: 2013
DOI: 10.1039/C3DT50551K
Abstract: The effect of human serum on the minimum inhibitory/bactericidal concentrations of the potential antimicrobial agents ΔΔ-[{Ru(phen)2}2(μ-bb(n))](4+) {ΔΔ-Rubb(n) where phen = 1,10-phenanthroline, bb(n) = 1,n-bis[4(4'-methyl-2,2'-bipyridyl)]-alkane for n = 12 and 16} against four strains of bacteria--Gram positive Staphylococcus aureus and methicillin-resistant S. aureus (MRSA), and Gram negative Escherichia coli and Pseudomonas aeruginosa--has been determined. The results demonstrated that the ruthenium(ii) complexes have significantly decreased in vitro activity in serum. Fluorescence spectroscopy was used to confirm that the decrease in antimicrobial activity was due to the strong binding of the ruthenium complexes with the serum proteins human serum albumin (HSA) and transferrin. A series of ruthenium complexes showed stronger binding to HSA than apo-transferrin but comparable or less than with holo-transferrin, with the binding affinity to all three proteins decreasing in the order trinuclear > dinuclear > mononuclear. The dinuclear complex ΔΔ-Rubb12 displaced warfarin from HSA, tentatively suggesting that the ruthenium complexes bind at or near the warfarin-binding site, Sudlow's site 1. The binding of ΔΔ-Rubb12 and ΔΔ-Rubb16 to the macrocyclic host molecule cucurbit[10]uril (Q[10]) was examined by NMR spectroscopy. The large upfield (1)H NMR chemical shift changes observed for the methylene protons in the bridging ligands upon addition of Q[10], coupled with the observation of a range of intermolecular ROEs in ROESY spectra, indicated that the dinuclear complexes bound Q[10] with the bridging ligand within the cavity and the metal centres positioned outside the portals. NMR and fluorescence spectroscopy demonstrated that the Q[10]-encapsulated ruthenium complexes directly bound HSA, and with similar affinity to the corresponding free metal complexes.
Publisher: Elsevier BV
Date: 11-2015
DOI: 10.1016/J.PREVETMED.2015.10.005
Abstract: There are few publications on brucellosis within the Pacific Island Countries and Territories (PICTs). The reason is possibly because the cattle population has been reportedly free of the disease for many years until a re-emergence occurred in the Fiji Islands (Viti Levu) in 2009. This paper reports on the outbreak of brucellosis in Fiji and its progression between 2009 and 2013 in the context of an overview of brucellosis in the Pacific Island community. Review of the literature found only 28 articles with the oldest record of brucellosis being in 1965 in Papua New Guinea (PNG) and from human cases in Tonga in 1980. The Fiji outbreak of Brucella abortus occurred in cattle in 2009 (Wainivesi basin) in the Tailevu province. Prior to the outbreak, Fiji declared freedom from B. abortus to OIE in 1996 after a successful eradication c aign. During the course of the outbreak investigation, serum s les were collected from between 9790 and 21,624 cattle per annum between 2009 and 2013 from 87 farms on the main island of Fiji (Viti Levu). Blood s les were tested for brucellosis using the Rose Bengal Test (RBT) in 2009 and the indirect ELISA test in subsequent years. At the time of the outbreak in Fiji (2009) the apparent prevalence in cattle was 1.50% and this has fluctuated since the outbreak. The True Prevalence (TP) for the main island in Fiji for the indirect ELISA tests was 2.40% in 2010, reached a peak of 3.49% in 2011 then reduced to 0.12% by 2013. The significant reduction in prevalence compared to 2010 is most likely due to the control programs being implemented in Fiji. The re-emergence of B. abortus in Fiji could be attributed to the lack of monitoring for the disease until 2009 combined with inadequate management of exposed animals, thus illustrating how important it is for authorities not to become complacent. Continued awareness and monitoring for brucellosis is essential if future outbreaks are to be avoided.
Publisher: Public Library of Science (PLoS)
Date: 19-07-2011
Publisher: Elsevier BV
Date: 07-2001
DOI: 10.1016/S1286-4579(01)01417-4
Abstract: Clinical presentations of melioidosis, caused by Burkholderia pseudomallei are protean, but the mechanisms underlying development of the different forms of disease remain poorly understood. In murine melioidosis, the level of virulence of B. pseudomallei is important in disease pathogenesis and progression. In this study, we used B. pseudomallei-susceptible BALB/c mice to determine the virulence of a library of clinical and environmental B. pseudomallei isolates from Australia and Papua New Guinea. Among 42 non-arabinose-assimilating (ara(-)) isolates, LD(50) ranged from 10 to > 10(6) CFU. There were numerous correlations between virulence and disease presentation in patients however, this was not a consistent observation. Virulence did not correlate with isolate origin (i.e. clinical vs environmental), since numerous ara(-) environmental isolates were highly virulent. The least virulent isolate was a soil isolate from Papua New Guinea, which was arabinose assimilating (ara(+)). Stability of B. pseudomallei virulence was investigated by in vivo passage of isolates through mice and repetitive in vitro subculture. Virulence increased following in vivo exposure in only one of eight isolates tested. In vitro subculture on ferric citrate-containing medium caused attenuation of virulence, and this correlated with changes in colony morphology. Pulsed-field gel electrophoresis and randomly lified polymorphic DNA typing demonstrated that selected epidemiologically related isolates that had variable clinical outcomes and different in vivo virulence were clonal strains. No molecular changes were observed in isolates after in vivo or in vitro exposure despite changes in virulence. These results indicate that virulence of selected B. pseudomallei isolates is variable, being dependent on factors such as iron bioavailability. They also support the importance of other variables such as inoculum size and host risk factors in determining the clinical severity of melioidosis.
Publisher: Oxford University Press (OUP)
Date: 08-2007
DOI: 10.1016/J.TRSTMH.2007.02.024
Abstract: A prospective study was conducted to determine the significance of melioidosis in the Balimo district of Western Province, Papua New Guinea. During 1998, after the establishment of laboratory procedures and increasing local clinical awareness, the disease was found in 1.8% (95% CI 0.37-5.1%) of in iduals presenting with fever refractory to standard treatment. The clinical incidence was 20.0 per 100,000 population (95% CI 12.2-30.9). The median age of culture-confirmed cases was 9.5 years (interquartile range 8.3-14.8 years). The seroprevalence of 747 community children in the region tested was 8.2% (95% CI 6.2-10.4%). Most in iduals presented during the rainy season with a febrile disease refractory to standard treatment, sometimes mimicking tuberculosis. Some family clustering was apparent. All patients with bacteraemic melioidosis died, but treatment with the available conventional therapies of chlor henicol, co-trimoxazole or doxycycline resulted in survival and cure in six patients with subacute/localised melioidosis. Further studies are needed to ascertain the local epidemiology and why children appear particularly at risk, as well as to establish the true extent of melioidosis in Papua New Guinea.
Publisher: Public Library of Science (PLoS)
Date: 14-09-2017
Publisher: Springer Singapore
Date: 2018
Publisher: Oxford University Press (OUP)
Date: 03-08-2012
DOI: 10.1093/JAC/DKS291
Abstract: To determine the in vitro susceptibility and cellular uptake for a series of dinuclear ruthenium(II) complexes [{Ru(phen)(2)}(2){μ-bb(n)}](4+) (Rubb(n)), and the mononuclear complexes [Ru(Me(4)phen)(3)](2+) and [Ru(phen)(2)(bb(7))](2+) against Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli and Pseudomonas aeruginosa. The in vitro susceptibility was determined by MIC and MBC assays, and time-kill curve experiments, while the cellular uptake was evaluated by monitoring the fluorescence of the complexes remaining in the supernatant of the cultures after incubation for various periods of time, flow cytometry and confocal microscopy. Rubb(12) and Rubb(16) are highly active, with MIC and MBC values of 1-2 mg/L (0.5-1 μM) for the two Gram-positive strains and 2-4 mg/L for E. coli and 16-32 mg/L for P. aeruginosa. Rubb(16) showed equal or better activity (on a molar basis) to gentamicin and icillin for all strains apart from P. aeruginosa. The relative MBC to MIC values indicated that Rubb(12) and Rubb(16) are bactericidal, and from the time-kill curve experiments, the ruthenium complexes can kill the bacteria within 2-6 h. The cellular uptake studies demonstrated that the observed antimicrobial activity is correlated with the level of uptake of the ruthenium complexes. Confocal microscopy confirmed the cellular uptake of Rubb(16), and tentatively suggested that the ruthenium complex is localized in the bacteria. The inert dinuclear ruthenium(II) complexes Rubb(12) and Rubb(16) have potential as new antimicrobial agents. The structure of the dinuclear ruthenium complexes can be readily further modified in order to increase their selectivity for bacteria over human cells.
Publisher: Frontiers Media SA
Date: 06-09-2017
Publisher: No publisher found
Date: 2014
DOI: 10.1016/J.ACTATROPICA.2013.12.017
Abstract: The Pacific Island countries and territories (PICTs) are reported to be free of the most serious infectious livestock diseases which are prevalent in other parts of the globe, such as Highly Pathogenic Avian Influenza, Foot and Mouth Disease or Rabies. Yet there is a lack of scientifically based evidence to confirm this animal health status. This paper reviews what has been published on diseases of domestic animals in the Pacific Islands region with a particular focus on data from the last 20 years (1992-2012). Relevant published papers were identified by a computerized literature search of two electronic databases (PubMed and Web of Knowledge). The latest reports on the animal health situation submitted by the PICTs to the World Organisation for Animal Health (OIE) were accessed on the World Animal Health Information Database (WAHID) interface and included in this review. Additionally, paper searches of resources were undertaken at the library of the Secretariat of the Pacific Community (SPC) in Fiji to retrieve any relevant grey literature for this review. The study eligibility criteria included qualitative or quantitative information on any disease (bacterial, viral, parasitic and other health disorders) affecting domestic terrestrial animals (mammals, reptiles, birds and bees) in any of the 22 PICTs members of the SPC. A total of 158 eligible references were retrieved of which only 77 (48.7%) were published since 1992 and analysed in more details. One hundred and one diseases and pathogens were reported on for bee, bird, carabao, cat, cattle, crocodile, deer, dog, donkey, goat, horse, pig, pigeon, poultry and sheep in the Oceania region and in 17 PICTs in particular. The paper gives information about known animal diseases, their reported prevalence and diseases not reported within the Pacific Islands region. The study found retrieved literature on animal diseases in PICTs was scarce and no longer up to date. There is a need to improve the published knowledge on the current animal disease status in the region.
Publisher: AIP Publishing
Date: 20-05-2013
DOI: 10.1063/1.4807438
Abstract: We introduce a chiral metamaterial with strong, non-resonant optical activity and very low polarization ellipticity. We achieve this by combining a meta-atom and its complementary structure into a meta-molecule, resulting in the coupling of magnetic and electric dipole responses. In contrast to either a pair of crosses or complementary crosses, this structure has low dispersion in the optical activity at the transmission resonance. We also study the excitation mechanism in this structure and optimize the optical activity through changing the twist angle.
Publisher: Oxford University Press (OUP)
Date: 30-11-2010
Publisher: MDPI AG
Date: 29-01-2023
DOI: 10.3390/MICROORGANISMS11020336
Abstract: Reference genes are frequently used for the normalization of quantitative reverse transcriptase PCR (qRTPCR) data in gene expression studies. Staphylococcus aureus is one of the most common causes of biofilm-related infections. Savirin and ticagrelor show in vitro as well as in vivo antibiofilm activity against S. aureus. The main aim of this study was to identify the most stably expressed reference genes to study the effect of these molecules on genes in a strong biofilm producing S. aureus isolate isolated from biofilm-related infection. Quantitative real-time PCR was performed by using relative quantification method. Four different algorithms, delta Ct, normfinder, bestkeeper, and genorm, followed by a comprehensive analysis was used to identify the most stable reference genes from a list of sixteen different candidate reference genes. All four algorithms reported different results, with some comparable findings among some methods. In the comprehensive analysis of the results of all the algorithms used, the most stable reference genes found were spa, rpoD, and pyk for savirin treatment experiment and gapdH, gyrA, and gmk for ticagrelor treatment experiment. The optimal number of reference genes required was two for both the experimental conditions. Despite having some drawbacks, each algorithm can reliably determine an appropriate reference gene independently. However, based on consensus ranking and the required optimal number of reference genes reported, spa and rpoD were the most appropriate reference genes for savirin treatment experiment, and gapdH and gyrA were most appropriate for ticagrelor treatment experiment. This study provides baseline data on reference genes to study the effect of savirin or ticagrelor treatment on the expression of potential reference genes in S. aureus. We recommend prior re-validation of reference genes on a case-by-case basis before they can be used.
Publisher: Royal Society of Chemistry (RSC)
Date: 2014
DOI: 10.1039/C4DT02139H
Abstract: Tri- and tetra-nuclear polypyridylruthenium( ii ) complexes have been synthesised which exhibit high levels of antimicrobial activity.
Publisher: Cambridge University Press (CUP)
Date: 22-08-2007
DOI: 10.1017/S0950268807009429
Abstract: The distribution of Burkholderia pseudomallei was determined in soil collected from a rural district in Papua New Guinea (PNG) where melioidosis had recently been described, predominately affecting children. In 274 s les, 2·6% tested culture-positive for B. pseudomallei . Pulsed-field gel electrophoresis using Spe I digests and rapid polymorphic DNA PCR with five primers demonstrated a single clone amongst clinical isolates and isolates cultured from the environment that was commonly used by children from whom the clinical isolates were derived. We concluded that in iduals in this region most probably acquired the organism through close contact with the environment at these sites. Burkholderia thailandensis , a closely related Burkholderia sp. was isolated from 5·5% of s les tested, an observation similar to that of melioidosis-endemic areas in Thailand. This is the first report of an environmental reservoir for melioidosis in PNG and confirms the Balimo district in PNG as melioidosis endemic.
Publisher: Public Library of Science (PLoS)
Date: 23-09-2015
Publisher: Public Library of Science (PLoS)
Date: 31-03-2011
Publisher: American Society for Microbiology
Date: 07-2015
DOI: 10.1128/AEM.00317-15
Abstract: Burkholderia pseudomallei is a saprophytic bacterium that causes melioidosis and is often isolated from rice fields in Southeast Asia, where the infection incidence is high among rice field workers. The aim of this study was to investigate the relationship between this bacterium and rice through growth experiments where the effect of colonization of domestic rice ( Oryza sativa L. cv Amaroo) roots by B. pseudomallei could be observed. When B. pseudomallei was exposed to surface-sterilized seeds, the growth of both the root and the aerosphere was retarded compared to that in controls. The organism was found to localize in the root hairs and endodermis of the plant. A biofilm formed around the root and root structures that were colonized. Growth experiments with a wild rice species ( Oryza meridionalis ) produced similar retardation of growth, while another domestic cultivar ( O. sativa L. cv Koshihikari) did not show retarded growth. Here we report B. pseudomallei infection and inhibition of O. sativa L. cv Amaroo, which might provide insights into plant interactions with this important human pathogen.
Publisher: Wiley
Date: 24-09-2012
Publisher: Public Library of Science (PLoS)
Date: 18-05-2012
Publisher: Elsevier BV
Date: 04-2009
Abstract: Sago starch is an important dietary carbohydrate in lowland Papua New Guinea (PNG). An investigation was conducted to determine whether microbes play a role in its preservation using traditional methods. In 12 stored sago s les collected from PNG villages, lactic acid bacteria (LAB) were present (> or = 3.6 x 10(4)cfu/g) and pH ranged from 6.8 to 4.2. Acetic and propionic acids were detected in all s les, while butyric, lactic and valeric acids were present in six or more. In freshly prepared sago, held in sealed containers in the laboratory at 30 degrees C, spontaneous fermentation by endogenous microflora of sago starch was observed. This was evident by increasing concentrations of acetic, butyric and lactic acids over 4 weeks, and pH reducing from 4.9 to 3.1: both LAB and yeasts were involved. Survival of potential bacterial pathogens was monitored by seeding sago starch with approximately 10(4)/g of selected organisms. Numbers of Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus fell to < 30/g within 7 days. Salmonella sp. was present only in low numbers after 7 days ( 10(2)/g). Fermentation appeared to increase the storability and safety of the product.
Publisher: Springer Science and Business Media LLC
Date: 12-04-2019
DOI: 10.1038/S41598-019-42211-Z
Abstract: Nanocomposites offer attractive and cost-effective thin layers with superior properties for antimicrobial, drug delivery and microelectronic applications. This work reports single-step plasma-enabled synthesis of polymer/zinc nanocomposite thin films via co-deposition of renewable geranium essential oil-derived polymer and zinc nanoparticles produced by thermal decomposition of zinc acetylacetonate. The chemical composition, surfaces characteristics and antimicrobial performance of the designed nanocomposite were systematically investigated. XPS survey proved the presence of ZnO in the matrix of formed polymers at 10 W and 50 W. SEM images verified that the average size of a ZnO nanoparticle slightly increased with an increase in the power of deposition, from approximately 60 nm at 10 W to approximately 80 nm at 50 W. Confocal scanning laser microscopy images showed that viability of S. aureus and E.coli cells significantly reduced on surfaces of ZnO olymer composites compared to pristine polymers. SEM observations further demonstrated that bacterial cells incubated on Zn/Ge 10 W and Zn/Ge 50 W had deteriorated cell walls, compared to pristine polymers and glass control. The release of ZnO nanoparticles from the composite thin films was confirmed using ICP measurements, and can be further controlled by coating the film with a thin polymeric layer. These eco-friendly nanocomposite films could be employed as encapsulation coatings to protect relevant surfaces of medical devices from microbial adhesion and colonization.
Publisher: Inter-Research Science Center
Date: 14-09-2007
DOI: 10.3354/DAO01838
Abstract: Mounting evidence implicates the disease chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis, in global hibian declines and extinctions. While the virulence of this disease has been clearly demonstrated, there is, as yet, no mechanistic explanation for how B. dendrobatidis kills hibians. To investigate the pathology of chytridiomycosis, blood s les were collected from uninfected, aclinically infected and clinically diseased hibians and analyzed for a wide range of biochemical and hematological parameters. Here, we show that green tree frogs Litoria caerulea with severe chytridiomycosis had reduced plasma osmolality, sodium, potassium, magnesium and chloride concentrations. Stable plasma albumin, hematocrit and urea levels indicated that hydration status was unaffected, signifying depletion of electrolytes from circulation rather than dilution due to increased water uptake. We suggest that B. dendrobatidis kills hibians by disrupting normal epidermal functioning, leading to osmotic imbalance through loss of electrolytes. Determining how B. dendrobatidis kills hibians is fundamental to understanding the host-pathogen relationship and thus the population declines attributed to B. dendrobatidis. Understanding the mechanisms of mortality may also explain interspecific variation in susceptibility to chytridiomycosis.
Publisher: Oxford University Press (OUP)
Date: 10-2008
Publisher: Public Library of Science (PLoS)
Date: 13-12-2011
Publisher: Oxford University Press (OUP)
Date: 08-12-2014
Abstract: The Balimo region in Papua New Guinea has previously been identified as melioidosis-endemic with a predilection for children. Where health resources are scarce, seroepidemiology can be used to assess exposure to Burkholderia pseudomallei and therefore risk of acquiring melioidosis. Logistic regression was used to determine associations between indirect haemagglutination assay (IHA) seroreactivity with environmental and demographic/cultural factors to aid in determining risk factors associated with exposure to B. pseudomallei in children. Of the 968 participants, 92.9% (899/968) were children, representing the majority of the community school population in the immediate Balimo region. Of these, 24.6% (221/899) were seropositive. Bathing in the lagoon (OR=2.679), drinking from the well or lagoon (OR=1.474), and being a member of the Siboko (OR=1.914) or Wagumisi (OR=1.942) clans were significantly associated with seropositivity. In the multivariate analysis, drinking from a well or lagoon (OR=1.713), and the Siboko (OR=2.341) and Wabadala (OR=2.022) clans were associated with seropositivity. This study in children supports observations that interactions with groundwater in this region are risk factors in acquiring melioidosis. Public health measures intended to limit this exposure may help reduce the risk of acquiring melioidosis in this remote community. Associations with clan structure may provide more cultural specific insights, however this requires further elucidation.
Publisher: American Society for Microbiology
Date: 08-2013
DOI: 10.1128/JCM.00418-13
Abstract: It has been hypothesized that biogeographical boundaries are a feature of Burkholderia pseudomallei ecology, and they impact the epidemiology of melioidosis on a global scale. This study examined the relatedness of B. pseudomallei sourced from islands in the Torres Strait of Northern Australia to determine if the geography of isolated island communities is a determinant of the organisms' dispersal. Environmental s ling on Badu Island in the Near Western Island cluster recovered a single clone. An additional 32 clinical isolates from the region were sourced. Isolates were characterized using multilocus sequence typing and a multiplex PCR targeting the flagellum gene cluster. Gene cluster analysis determined that 69% of the isolates from the region encoded the ancestral Burkholderia thailandensis -like flagellum and chemotaxis gene cluster, a proportion significantly lower than that reported from mainland Australia and consistent with observations of isolates from southern Papua New Guinea. A goodness-of-fit test indicated that there was geographic localization of sequence types throughout the archipelago, with the exception of Thursday Island, the economic and cultural hub of the region. Sequence types common to mainland Australia and Papua New Guinea were identified. These findings demonstrate for the first time an environmental reservoir for B. pseudomallei in the Torres Strait, and multilocus sequence typing suggests that the organism is not randomly distributed throughout this region and that seawater may provide a barrier to dispersal of the organism. Moreover, these findings support an anthropogenic dispersal hypothesis for the spread of B. pseudomallei throughout this region.
Publisher: Royal Society of Chemistry (RSC)
Date: 2013
DOI: 10.1039/C3DT32775B
Abstract: A series of polypyridyl-ruthenium(II) and -iridium(III) complexes that contain labile chlorido ligands, [{M(tpy)Cl}(2){μ-bb(n)}](2/4+) {Cl-Mbb(n) where M = Ru or Ir tpy = 2,2':6',2''-terpyridine and bb(n) = bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (n = 7, 12 or 16)} have been synthesised and their potential as antimicrobial agents examined. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the series of metal complexes against four strains of bacteria - Gram positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), and Gram negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) - have been determined. All the ruthenium complexes were highly active and bactericidal. In particular, the Cl-Rubb(12) complex showed excellent activity against all bacterial cell lines with MIC values of 1 μg mL(-1) against the Gram positive bacteria and 2 and 8 μg mL(-1) against E. coli and P. aeruginosa, respectively. The corresponding iridium(III) complexes also showed significant antimicrobial activity in terms of MIC values however and surprisingly, the iridium complexes were bacteriostatic rather than bactericidal. The inert iridium(III) complex, [{Ir(phen)(2)}(2){μ-bb(12)}](6+) {where phen = 1,10-phenanthroline) exhibited no antimicrobial activity, suggesting that it could not cross the bacterial membrane. The mononuclear model complex, [Ir(tpy)(Me(2)bpy)Cl]Cl(2) (where Me(2)bpy = 4,4'-dimethyl-2,2'-bipyridine), was found to aquate very rapidly, with the pK(a) of the iridium-bound water in the corresponding aqua complex determined to be 6.0. This suggests the dinuclear complexes [Ir(tpy)Cl}(2){μ-bb(n)}](4+) aquate and deprotonate rapidly and enter the bacterial cells as 4+ charged hydroxo species.
Publisher: American Society for Microbiology
Date: 26-02-2015
Abstract: Here, we present the draft genome sequences of 80 isolates of Burkholderia pseudomallei . The isolates represent clinical cases of melioidosis and environmental isolates from regions in Australia and Papua New Guinea where B. pseudomallei is endemic. The genomes provide further context for the ersity of the pathogen.
Publisher: Springer Science and Business Media LLC
Date: 02-09-2010
DOI: 10.1007/S11046-009-9235-3
Abstract: Sago haemolytic disease (SHD) is a rare but often fatal illness linked to consumption of stale sago starch in Papua New Guinea. Although the aetiology of SHD remains unknown, mycotoxins are suspected. This study investigated whether fungi isolated from Papua New Guinean sago starch were haemolytic. Filamentous fungi and yeasts from sago starch were grown on sheep blood agar and some on human blood agar. Clear haemolytic activity was demonstrated by 55% of filamentous fungal isolates, but not by yeasts. A semi-quantitative bioassay was developed involving incubation of human erythrocytes with fungal extracts. Extracts of cultures of Penicillium, Aspergillus and Fusarium all caused rapid haemolysis in the bioassay. Partial fractionation of extracts suggested that both polar and non-polar haemolytic components had haemolytic activity in vitro. Further work is warranted to identify these metabolites and determine if they play a role in SHD.
Publisher: Springer Singapore
Date: 2018
Publisher: Public Library of Science (PLoS)
Date: 05-11-2018
Publisher: Springer Science and Business Media LLC
Date: 30-11-2015
DOI: 10.1007/S12223-015-0438-3
Abstract: Burkholderia pseudomallei is the environmental bacterium that causes the serious disease melioidosis. Recently, a high prevalence of viable B. pseudomallei was reported from natural groundwater seeps around Castle Hill, a clinical focus of melioidosis in Townsville, Australia. This study sought to expand previous findings to determine the extent of B. pseudomallei in more erse natural groundwater seeps in northern Queensland to ascertain if the presence of the organism in groundwater on Castle Hill was an isolated occurrence. Analysis of water s les (n = 26) obtained from natural groundwater seeps following an intensive rainfall event in the Townsville region determined the presence of B. pseudomallei DNA in duplicates of 18 s les (69.2 % [95 % CI, 51.5 to 87.0]). From 26 water s les, a single isolate of B. pseudomallei was recovered despite plating of both pre-enriched s les and original water s les onto selective media, indicating that the sensitivity of these molecular techniques far exceeds culture-based methods. Furthermore, the identification of new environments endemic for melioidosis may be more effectively determined by analysing surface groundwater seeps than by the analysis of random soil s les. This study suggests that a higher incidence of melioidosis following monsoonal rains may be partially the result of exposure to groundwater sources carrying B. pseudomallei, and that modifications to public health messages in endemic regions may be warranted. Moreover, these findings have implications for predictive models of melioidosis, effective models requiring consideration of topographical and surface hydrological data.
Publisher: American Society for Microbiology
Date: 15-10-2011
DOI: 10.1128/AEM.05048-11
Abstract: Burkholderia pseudomallei is a saprophytic bacterium which is the causative agent of melioidosis, a common cause of fatal bacterial pneumonia and sepsis in the tropics. The incidence of melioidosis is clustered spatially and temporally and is heavily linked to rainfall and extreme weather events. Clinical case clustering has recently been reported in Townsville, Australia, and has implicated Castle Hill, a granite monolith in the city center, as a potential reservoir of infection. Topsoil and water from seasonal groundwater seeps were collected around the base of Castle Hill and analyzed by quantitative real-time PCR targeting the type III secretion system genes for the presence of B. pseudomallei . The organism was identified in 65% (95% confidence interval [CI], 49.5 to 80.4) of soil s les ( n = 40) and 92.5% (95% CI, 83.9 to 100) of seasonal groundwater s les ( n = 40). Further s ling of water collected from roads and gutters in nearby residential areas after an intense rainfall event found that 88.2% (95% CI, 72.9 to 100) of s les ( n = 16) contained viable B. pseudomallei at concentrations up to 113 CFU/ml. Comparison of isolates using multilocus sequence typing demonstrated clinical matches and close associations between environmental isolates and isolates derived from clinical s les from patients in Townsville. This study demonstrated that waterborne B. pseudomallei from groundwater seeps around Castle Hill may facilitate exposure to B. pseudomallei and contribute to the clinical clustering at this site. Access to this type of information will advise the development and implementation of public health measures to reduce the incidence of melioidosis.
Publisher: Elsevier BV
Date: 03-2015
DOI: 10.1016/J.ACTATROPICA.2014.12.012
Abstract: Most Pacific Island countries and territories (PICTs) have developing economies and face a critical shortage of veterinarians with limited financial resources allocated to their animal disease surveillance programmes. Thus, animal health authorities have to set priorities for better focusing their scarce resources. The main objective of this study was to identify animal diseases perceived to be of importance by decision makers within selected PICTs, at the regional and national levels, to ensure better targeting of animal health resources. A second objective was to investigate whether the targeted surveillance programmes resulting from this rationalized approach would also benefit the local communities engaged in livestock production. A multi-criteria prioritization process was developed, involving local experts, to score and rank 132 animal diseases based on their priority at the regional and national levels for four PICTs: Fiji, Papua New Guinea, Solomon Islands, and Vanuatu, which form part of a regional Food Animal Biosecurity Network. In parallel interviews with farmers and field animal health and production workers were conducted to assess their perception of animal diseases. The list of the top-twenty ranked diseases for the Pacific Islands region shows a mix of endemic zoonotic diseases (such as leptospirosis ranked first brucellosis third tuberculosis sixth and endoparasites and ectoparasites, respectively eleventh and thirteenth) with exotic diseases (such as HPAI ranked second, FMD fifth, and rabies ninth). There were different disease ranking lists for each of the four targeted PICTs, confirming different strategies of disease prevention and control may be required for each country, rather than a regional approach. Interviewed animal health and production workers were unfamiliar with most of the prioritized diseases and a majority acknowledged that they would not be able to recognize clinical signs if outbreaks were to occur in their area. Leptospirosis, which is endemic and identified as the top priority disease at the regional level, was never mentioned by any interviewed farmer. Farmers did not name any emerging infectious diseases as priorities. Instead, they identified endemic diseases (parasites, flu, coccidiosis, and scabies) as the most important. While animal disease priorities appear to differ widely between the targeted regions and countries, it also varies significantly between experts and farmers. Better targeted surveillance programmes may thus result in more rational and transparent allocation of resources, and thus enhanced food security, but may not directly match the needs of the local communities.
Publisher: Elsevier BV
Date: 02-2018
DOI: 10.1016/J.IJID.2017.12.004
Abstract: Tuberculosis (TB) is a serious health problem in Papua New Guinea (PNG) with an estimated 30000 new cases and 3800 deaths each year. In the Balimo region of the Western Province, diagnosis relies on clinical manifestations and on the microscopic detection of acid-fast bacilli (AFB) in sputum smears, a technique with limited sensitivity. A molecular diagnosis assay targeting DNA extracted from archived sputum smear slides collected from the Balimo region (2012-2014) was conducted, without the need for a viable culture. The presence of Mycobacterium sp on 1162 slides prepared from 345 sputum s les was assessed using a real-time PCR (qPCR) approach. The qPCR technique identified the presence of mycobacteria in 35.4% of the smear slides and 59.7% of the tested sputum s les. Poor agreement was observed between the two diagnosis methods (smear AFB microscopy versus qPCR), with 100 AFB-positive sputum s les compared to 206 qPCR-positive sputum s les overall. Treatment was initiated in 90.2% of the smear-positive cases. Unnecessary treatment of 'false-positive' TB cases (AFB-negative/qPCR-negative) was very low (8.6%) and was even lower when the nine patients diagnosed with extrapulmonary TB were excluded from the analysis. However, the prevalence of false-negatives (AFB-negative/qPCR-positive) was high (28.5%). Undetected smear-negative TB is occurring in the Balimo region of PNG, as well as some unnecessary empirical treatment. Molecular methods of diagnosis could greatly reduce the frequency of inappropriate clinical assessment, as well as providing point-of-care diagnosis. This may provide substantial patient and programmatic benefits, including lowering the economic burden on patients from rural areas seeking medical diagnosis in Balimo.
Publisher: MDPI AG
Date: 07-04-2017
Publisher: Springer Science and Business Media LLC
Date: 13-01-2017
Publisher: American Society for Microbiology
Date: 06-2014
DOI: 10.1128/AEM.00128-14
Abstract: Burkholderia pseudomallei is a Gram-negative soil bacillus that is the etiological agent of melioidosis and a biothreat agent. Little is known about the biogeography of this bacterium in Australia, despite its hyperendemicity in the northern region of this continent. The population structure of 953 Australian B. pseudomallei strains representing 779 and 174 isolates of clinical and environmental origins, respectively, was analyzed using multilocus sequence typing (MLST). Bayesian population structure and network SplitsTree analyses were performed on concatenated MLST loci, and sequence type (ST) ersity and evenness were examined using Simpson's and Pielou's indices and a multivariate dissimilarity matrix. Bayesian analysis found two B. pseudomallei populations in Australia that were geographically distinct isolates from the Northern Territory were grouped mainly into the first population, whereas the majority of isolates from Queensland were grouped in a second population. Differences in ST evenness were observed between s ling areas, confirming that B. pseudomallei is widespread and established across northern Australia, with a large number of fragmented habitats. ST analysis showed that B. pseudomallei populations ersified as the s ling area increased. This observation was in contrast to smaller s ling areas where a few STs predominated, suggesting that B. pseudomallei populations are ecologically established and not frequently dispersed. Interestingly, there was no identifiable ST bias between clinical and environmental isolates, suggesting the potential for all culturable B. pseudomallei isolates to cause disease. Our findings have important implications for understanding the ecology of B. pseudomallei in Australia and for potential source attribution of this bacterium in the event of unexpected cases of melioidosis.
Publisher: MDPI AG
Date: 11-04-2017
Publisher: MDPI AG
Date: 13-09-2022
Abstract: Whole-blood-derived transcriptional profiling is widely used in biomarker discovery, immunological research, and therapeutic development. Traditional molecular and high-throughput transcriptomic platforms, including molecular assays with quantitative PCR (qPCR) and RNA-sequencing (RNA-seq), are dependent upon high-quality and intact RNA. However, collecting high-quality RNA from field studies in remote tropical locations can be challenging due to resource restrictions and logistics of post-collection processing. The current study tested the relative performance of the two most widely used whole-blood RNA collection systems, PAXgene® and Tempus™, in optimal laboratory conditions as well as suboptimal conditions in tropical field sites, including the effects of extended storage times and high storage temperatures. We found that Tempus™ tubes maintained a slightly higher RNA quantity and integrity relative to PAXgene® tubes at suboptimal tropical conditions. Both PAXgene® and Tempus™ tubes gave similar RNA purity (A260/A280). Additionally, Tempus™ tubes preferentially maintained the stability of mRNA transcripts for two reference genes tested, Succinate dehydrogenase complex, subunit A (SDHA) and TATA-box-binding protein (TBP), even when RNA quality decreased with storage length and temperature. Both tube types preserved the rRNA transcript 18S ribosomal RNA (18S) equally. Our results suggest that Tempus™ blood RNA collection tubes are preferable to PAXgene® for whole-blood collection in suboptimal tropical conditions for RNA-based studies in resource-limited settings.
Publisher: MDPI AG
Date: 15-03-2018
Publisher: Public Library of Science (PLoS)
Date: 21-03-2013
Publisher: Wiley
Date: 05-07-2018
DOI: 10.1111/TMI.13118
Abstract: Papua New Guinea (PNG) has an emerging tuberculosis (TB) epidemic which has become a national public health priority. In Western Province, there are few data about TB outside Daru and the South Fly District. This study describes the epidemiology of TB diagnosed at Balimo District Hospital (BDH) in the Middle Fly District of Western Province, PNG. All patients (n = 1614) diagnosed with TB at BDH from April 2013 to February 2017 were recorded. Incidence of reported new cases was calculated for the combined Balimo Urban and Gogodala Rural local level government areas. Analyses investigated patient demographic and clinical information, differences between pulmonary and extrapulmonary TB patients, and predictors of treatment failure. The average case notification rate (2014-2016) was 727 TB cases per 100 000 people per year. One-quarter of TB cases were in children, and 77.1% of all cases had an extrapulmonary TB diagnosis. There was a 1:1.1 ratio of female to male TB cases. When comparing pulmonary and extrapulmonary TB patients, extrapulmonary TB was more likely in those aged up to 14 years and over 54 years. Extrapulmonary TB was more likely in new patients, and pulmonary TB more likely in previously treated patients. Residence in rural regions was associated with treatment failure. There is a high burden of TB in the Balimo region, including a very high proportion of extrapulmonary TB. These factors emphasise the importance of BDH as the primary hospital for TB cases in the Balimo region and the Middle Fly District, and the need for resources and staff to manage both drug-susceptible and drug-resistant TB cases.
Publisher: Elsevier BV
Date: 05-2010
Publisher: Royal Society of Chemistry (RSC)
Date: 2011
DOI: 10.1039/C1DT10250H
Abstract: The minimum inhibitory concentrations (MIC) of a series of synthetic inert polypyridylruthenium(II) complexes against four strains of bacteria--Gram positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), and Gram negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa)--have been determined. The results demonstrate that for the dinuclear ruthenium(II) complexes ΔΔ/ΛΛ-[{Ru(phen)(2)}(2){μ-bb(n)}](4+) {where phen = 1,10-phenanthroline bb(n) = bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (n = 2, 5, 7, 10, 12 or 16)} the complexes linked by the bb(12), bb(14) and bb(16) ligands are highly active, with MIC values of 1 μg mL(-1) against both S. aureus and MRSA, and 2-4 and 8-16 μg mL(-1) against E. coli and P. aeruginosa, respectively. The mononuclear complex [Ru(Me(4)phen)(3)](2+) showed equal activity (on a mole basis) against S. aureus compared with the Rubb(12), Rubb(14) and Rubb(16), but was considerably less active against MRSA and the two Gram negative bacteria. For the dinuclear Rubb(n) family of complexes, the antimicrobial activity was related to the octanol-water partition coefficient (logP). However, the highly lipophilic mononuclear complex Δ-[Ru(phen)(2)(bb(16))](2+) was significantly less active than Rubb(16), highlighting the importance of the dinuclear structure. Preliminary toxicity assays were also carried out for the ΔΔ isomers of Rubb(7), Rubb(10), Rubb(12) and Rubb(16) against two human cells lines, fresh red blood cells and THP-1 cells. The results showed that the dinuclear ruthenium complexes are significantly less toxic to human cells compared to bacterial cells, with the HC(50) and IC(50) values 100-fold higher than the MIC for the complex that showed the best potential--ΔΔ-Rubb(12).
Publisher: Oxford University Press (OUP)
Date: 19-07-2013
DOI: 10.1093/JAC/DKT279
Abstract: To determine the energy dependency of and the contribution of the membrane potential to the cellular accumulation of the dinuclear complexes [{Ru(phen)2}2{μ-bbn}](4+) (Rubbn) and the mononuclear complexes [Ru(Me4phen)3](2+) and [Ru(phen)2(bb7)](2+) in Staphylococcus aureus and Escherichia coli, and to examine their effect on the bacterial membrane. The accumulation of the ruthenium complexes in bacteria was determined using flow cytometry at a range of temperatures. The cellular accumulation of the ruthenium complexes was also determined in cells that had been incubated with the metal complexes in the presence or absence of metabolic stimulators or inhibitors and/or commercial dyes to determine the membrane potential or membrane permeability. The accumulation of ruthenium complexes in the two bacterial strains was shown to increase with increasing incubation temperature, with the relative increase in accumulation greater with E. coli, particularly for Rubb12 and Rubb16. No decrease in accumulation was observed for Rubb12 in ATP-inhibited cells. While carbonyl cyanide m-chlorophenyl hydrazone (CCCP) did depolarize the cell membrane, no reduction in the accumulation of Rubb12 was observed however, all ruthenium complexes, when incubated with S. aureus at concentrations twice their MIC, depolarized the membrane to a similar extent to CCCP. Except for the mononuclear complex [Ru(Me4phen)3](2+), incubation of any of the other ruthenium complexes allowed a greater quantity of the membrane-impermeable dye TO-PRO-3 to be taken up by S. aureus. The results indicate that the potential new antimicrobial Rubbn complexes enter the cell in an energy-independent manner, depolarize the cell membrane and significantly permeabilize the cellular membrane.
Publisher: Royal Society of Chemistry (RSC)
Date: 2017
DOI: 10.1039/C7OB00724H
Abstract: The relatively non-toxic family of cucurbit[n]uril, Q[n], have shown considerable potential in vitro as drug delivery agents, with only a few ex les of pharmacokinetic (PK) studies for drug⊂Q[n]. Drug-free Q[n] PK studies are the next step in determining the pharmacological applicability in their drug delivery potential. The results for the first PK and bio-distribution of drug-free
Publisher: Oxford University Press (OUP)
Date: 05-03-2016
DOI: 10.1093/JAC/DKW026
Abstract: The objectives of this study were to: (i) determine the in vitro activities of a series of di-, tri- and tetra-nuclear ruthenium complexes (Rubbn, Rubbn-tri and Rubbn-tetra) against a range of Gram-positive and -negative bacteria and compare the antimicrobial activities with the corresponding toxicities against eukaryotic cells and (ii) compare MIC values with achievable in vivo serum concentrations for the least toxic ruthenium complex. The in vitro activities were determined by MIC assays and time-kill curve experiments, while the toxicities of the ruthenium complexes were determined using the Alamar blue cytotoxicity assay. A preliminary pharmacokinetic study was undertaken to determine the Rubb12 serum concentration in mice as a function of time after administration. Rubb12, Rubb12-tri and Rubb12-tetra are highly active, with MIC values of 1-2 mg/L (0.5-1.5 μM) for a range of Gram-positive strains, but showed variable activities against a panel of Gram-negative bacteria. Time-kill experiments indicated that Rubb12, Rubb12-tri and Rubb12-tetra are bactericidal and kill bacteria within 3-8 h. The di-, tri- and tetra-nuclear complexes were ∼50 times more toxic to Gram-positive bacteria and 25 times more toxic to Gram-negative strains, classified as susceptible, than to liver and kidney cells. Preliminary pharmacokinetic experiments established that serum concentrations higher than MIC values can be obtained for Rubb12 with an administered dose of 32 mg/kg. The ruthenium complexes, particularly Rubb12, have potential as new antimicrobial agents. The structure of the dinuclear ruthenium complex can be readily further modified in order to increase the selectivity for bacteria over eukaryotic cells.
Publisher: Springer Science and Business Media LLC
Date: 29-10-2019
DOI: 10.1038/S41598-019-51892-5
Abstract: Tuberculosis remains the world’s leading cause of death from an infectious agent, and is a serious health problem in Papua New Guinea (PNG) with an estimated 36,000 new cases each year. This study describes the genetic ersity of Mycobacterium tuberculosis among tuberculosis patients in the Balimo/Bamu region in the Middle Fly District of Western Province in PNG, and investigates rif icin resistance-associated mutations. Archived Ziehl-Neelsen-stained sputum smears were used to conduct microbead-based spoligotyping and assess genotypic resistance. Among the 162 s les included, 80 (49.4%) generated spoligotyping patterns (n = 23), belonging predominantly to the L2 Lineage (44%) and the L4 Lineage (30%). This is consistent with what has been found in other PNG regions geographically distant from Middle Fly District of Western Province, but is different from neighbouring South-East Asian countries. Rif icin resistance was identified in 7.8% of the successfully sequenced s les, with all resistant s les belonging to the L2/Beijing Lineage. A high prevalence of mixed L2/L4 profiles was suggestive of polyclonal infection in the region, although this would need to be confirmed. The method described here could be a game-changer in resource-limited countries where large numbers of archived smear slides could be used for retrospective (and prospective) studies of M. tuberculosis genetic epidemiology.
No related grants have been discovered for Jeffrey Warner.