The epidemiology of malaria in Kutubu, Southern Highlands Province, Papua New Guinea, before and during a private sector initiative for malaria control

Papua New Guinea (PNG) has a significant malaria burden, is resource constrained, and has isolated populations with limited access to health services. Home-based management is a key element of the national program that supports strategies of early detection, diagnosis and treatment. We describe the epidemiology of malaria near Lake Kutubu in the Southern Highlands Province through reported data on suspected and confirmed malaria in patients accessing public health facilities or using a novel, incentivised, social marketing approach for malaria treatment at the village level. Monthly case data reported by nine health facilities and 14 village-based providers, known as Marasin Stoa Kipas (MSK), were extracted from outpatient registers and MSK malaria case forms. Descriptive statistics of diagnostic use, monthly incidence, test positivity rate and species distribution were estimated. Summary statistics of service delivery demonstrate patient access and diagnostic coverage in program areas. From May 2005 to September 2013, 15,726 individuals were tested with either rapid diagnostic test and/or microscopy at health facilities, and 42% had a positive result for malaria (n= 6604); of these 67.1% (n=4431) were positive for P. falciparum (alone or mixed) and 32.9% were positive for non-P. falciparum species (alone or mixed). From October 2007 to September 2013, 9687 individuals were tested with either RDT and/or microscopy at MSK sites and 44.2% (n=4283) tested positive for malaria; of these, 65.3% (n=2796) were positive for P. falciparum, while 34.7% (n=1487) were positive for non-P. falciparum species. Up to April 2010 there was an intermittent and upward trend in the reported incidence of all species of confirmed malaria, reaching 50 per 1000 population per month for both sites combined, followed by a steady decline to four per 1000 population per month in 2013, with P. vivax the most common infection. This study is the most recent longitudinal overview of malaria in the Southern Highlands since 2003. It outlines patient access to a community-based model of care. The analysis shows changes in health facility versus MSK use, a strongly decreasing trend in incidence of confirmed malaria from 2010 to 2013, and a shift from predominantly P. falciparum to P. vivax infection

Macrophage activation in the presence of Burkholderia pseudomallei

Melioidosis is a potentially fatal disease caused by the soil dwelling bacterium Burkholderia pseudomallei. The disease is endemic in tropical and subtropical regions of the world but is primarily located in southeast Asia and in northern Australia. Acute melioidosis is characterised by a fulminating septicaemia that can result in death within days of exposure. The generation of sepsis results from a prolonged and or exaggerated stimulation of host immune cells by pathogens which culminates in the hyperproduction of inflammatory mediators. Stimulation of host cells by pathogens is facilitated by pattern recognition receptors which bind to microbial structures and initiate downstream signalling cascades. The most well studied pattern recognition receptors are those in the Toll-like receptor family (TLRs). Over the past decade a tremendous amount of work has been conducted to identify TLR specific ligands, TLR structures, associated signalling proteins and the cytokines produced by TLR activation. Understanding the nature of TLR mediated signalling in host cells is fundamental to elucidating the pathogenesis of sepsis and the improvement of clinical management. Up until 2007, no extensive publications had surfaced regarding TLR recognition of B. pseudomallei and the role of TLRs still remains an area of intense study. Therefore, the major focus of the research outlined within this thesis was the characterisation of TLR activation by B. pseudomallei during acute infection. This was achieved using infection studies in murine and human cell lines as well as in primary cells isolated from a previously characterised murine model of acute melioidosis. Primary cells were also isolated from partially resistant murine hosts and used in infection studies.\ud \ud To ascertain the degree of TLR activation by 26 B. pseudomallei isolates with varying levels of virulence, standard antibiotic protection assays were performed on RAW 264.7 macrophages and peritoneal exudate cells (PEC) challenged with B. pseudomallei. Reverse transcriptase-polymerase chain reaction (RT PCR) was performed to determine TLR2, TLR4, TLR5, and TLR9 expression. Internalisation and killing of bacteria were determined at the early stages of infection. ELISAs were performed to determine total protein levels of tumor necrosis factor alpha (TNF-α) from cultured supernatants. Griess assays were used to assess nitrite production by macrophages as a measure of cytotoxic activity. Up to 2 h post infection B. pseudomallei failed to significantly increase TLR4, TLR5 and TLR9 expression in both cell types. However, TLR2 expression was increased, irrespective of isolate virulence in RAW 264.7 macrophages. Levels of TNF-α and nitrite were significantly attenuated in RAW 264.7 macrophages and no correlation was found between the level of virulence of the infecting strain and TLR expression, bacterial uptake or killing. The ability of B. pseudomallei to evade detection by macrophages may be in part, due to possible signal dampening of TLR receptors at the early stages of infection.\ud \ud Susceptibility to B. pseudomallei infection is determined by host immunocompetence as well as bacterial virulence. During acute melioidosis, excessive levels of pro-inflammatory cytokines are found systemically and lead to fatal septicaemias. Using RT PCR analysis, we found that B. pseudomallei can induce TLR4, TLR5, and TLR9 expression in peritoneal exudate cells (PECs) derived from susceptible and partially resistant mice. Induction of TLR4, TLR5, and TLR9 expression, in addition to TNF-α and interleukin 12, p40 subunit (IL-12p40), was greater in susceptible hosts. These results indicate the importance of genetic factors in regards to TLR recognition and response to B. pseudomallei and indicate more pronounced TLR activation in susceptible hosts.\ud \ud To determine if macrophage activation is ubiquitous in the presence of B. pseudomallei isolates of different origin, we examined the role of TLR2 and TLR4 in the recognition of clinical isolates of high and low virulence. Using quantitative real time polymerase chain reaction (qRT PCR) analysis, transfection assays and ELISA, we determined transcription profiles of TLRs and cytokine secretion in macrophages co-cultured with B. pseudomallei. Our findings demonstrate that there are differences in TLR transcription profiles between isolates and that contrary to previous reports the degree of TLR2 and TLR4 mediated NF-κB activation may be dependant on the individual B. pseudomallei isolate.\ud \ud In summary, the results in the present study have provided a basic understanding of TLR involvement in B. pseudomallei recognition. They provide supporting evidence regarding the role of TLRs during melioidosis and the establishment of different TLR transcription in hosts with different susceptibilities to infection. These results also suggest that B. pseudomallei activation of TLRs may be different between isolates

The Epidemiology of Malaria in Kutubu, Southern Highlands Province, Papua New Guinea, before and during a Private Sector Initiative for Malaria Control

Papua New Guinea (PNG) has a significant malaria burden, is resource constrained, and has isolated populations with limited access to health services. Home-based management is a key element of the national program that supports strategies of early detection, diagnosis and treatment. We describe the epidemiology of malaria near Lake Kutubu in the Southern Highlands Province through reported data on suspected and confirmed malaria in patients accessing public health facilities or using a novel, incentivised, social marketing approach for malaria treatment at the village level. Monthly case data reported by nine health facilities and 14 village-based providers, known as Marasin Stoa Kipas (MSK), were extracted from outpatient registers and MSK malaria case forms. Descriptive statistics of diagnostic use, monthly incidence, test positivity rate and species distribution were estimated. Summary statistics of service delivery demonstrate patient access and diagnostic coverage in program areas. From May 2005 to September 2013, 15,726 individuals were tested with either rapid diagnostic test and/or microscopy at health facilities, and 42% had a positive result for malaria (n = 6604); of these 67.1% (n = 4431) were positive for P. falciparum (alone or mixed) and 32.9% were positive for non-P. falciparum species (alone or mixed). From October 2007 to September 2013, 9687 individuals were tested with either RDT and/or microscopy at MSK sites and 44.2% (n = 4283) tested positive for malaria; of these, 65.3% (n = 2796) were positive for P. falciparum, while 34.7% (n = 1487) were positive for non-P. falciparum species. Up to April 2010 there was an intermittent and upward trend in the reported incidence of all species of confirmed malaria, reaching 50 per 1000 population per month for both sites combined, followed by a steady decline to four per 1000 population per month in 2013, with P. vivax the most common infection. This study is the most recent longitudinal overview of malaria in the Southern Highlands since 2003. It outlines patient access to a community-based model of care. The analysis shows changes in health facility versus MSK use, a strongly decreasing trend in incidence of confirmed malaria from 2010 to 2013, and a shift from predominantly P. falciparum to P. vivax infection

The effect of different Burkholderia pseudomallei isolates of varying levels of virulence on toll-like-receptor expression

The purpose of this investigation was to ascertain the degree of toll-like-receptor (TLR) activation by Burkholderia pseudomallei isolates with varying levels of virulence 2 h post infection. Standard antibiotic protection assays were performed on RAW 264.7 macrophages and peritoneal exudate cells (PEC) challenged with B. pseudomallei. Real-time PCR (RT-PCR) was performed to determine TLR2, TLR4, TLR5 and TLR9 expression. Internalization and killing of bacteria were determined 2h post infection. ELISAs were performed to determine the levels of TNF-α from cultured supernatants. Nitrate levels were determined by Griess assays. Up to 2h post infection, B. pseudomallei failed to significantly increase TLR4, TLR5 and TLR9 expression in both cell types. However, TLR2 expression was increased in RAW 264.7 macrophages, irrespective of isolate virulence. The levels of TNF-α and nitrate were significantly attenuated in RAW 264.7 macrophages, and no correlation was found between the level of virulence of the infecting strain and TLR expression, bacterial uptake, or killing. The ability of B. pseudomallei to evade detection by macrophages may in part be due to possible signal dampening of TLRs at very early stages of infection

Activity of tigecycline in the treatment of acute Burkholderia pseudomallei infection in a murine model

Burkholderia pseudomallei is the causative agent of melioidosis. Standard therapy includes ceftazidime alone or in combination with co-trimoxazole. Tigecycline, a novel agent, has displayed activity against B. pseudomallei. We evaluated the in vivo efficacy of tigecycline using a murine model of melioidosis. Mice were infected with either a high or low virulence B. pseudomallei isolate followed by administration of antibiotics alone or in combination (tigecycline, ceftazidime, tigecycline plus ceftazidime) for 7 days. Bacterial loads were assessed up to 7 days and survival was determined up to 7 days post infection. Tigecycline in combination with ceftazidime was the most effective and conferred the lowest mortality, suggesting the use of this new agent in B. pseudomallei infection

Altered macrophage function is associated with severe Burkholderia pseudomallei infection in a murine model of type 2 diabetes

This study used a murine model of type 2 diabetes (BKS.Cg-Dock7m +/+ Leprdb/J mice) to investigate the inflammatory and cellular mechanisms predisposing to Burkholderia pseudomallei infection and co-morbid diabetes. Homozygous db/db (diabetic) mice developed extreme obesity, dyslipidaemia and glucose intolerance leading to hyperglycaemia and overt type 2 diabetes. Compared to their heterozygous db/+ (non-diabetic) littermates, diabetic mice rapidly succumbed to subcutaneous B. pseudomallei infection, paralleled by severe hypoglycaemia and increased expression of the proinflammatory cytokines, tumour necrosis factor (TNF)-α and interleukin (IL)-1β, in the spleen, despite comparable bacterial loads in the spleen of non-diabetic mice. Neutrophil oxidative burst and dendritic cell uptake and killing of B. pseudomallei were similar between diabetic and non-diabetic mice. Compared to peritoneal macrophages from non-diabetic mice, macrophages from diabetic mice were unable to contain and kill B. pseudomallei. Functional differences between macrophages of diabetic and non-diabetic mice toward B. pseudomallei may contribute to rapid dissemination and more severe disease progression in hosts with co-morbid type 2 diabetes

The antimicrobial activity of inert oligonuclear polypyridylruthenium(II) complexes against pathogenic bacteria, including MRSA

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-bbn}]4+ {where phen = 1,10-phenanthroline; bbn = bis[4(4′-methyl-2,2′-bipyridyl)]-1,n-alkane (n = 2, 5, 7, 10, 12 or 16)} the complexes linked by the bb12, bb14 and bb16 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(Me4phen)3]2+ showed equal activity (on a mole basis) against S. aureus compared with the Rubb12, Rubb14 and Rubb16, but was considerably less active against MRSA and the two Gram negative bacteria. For the dinuclear Rubbn 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(bb16)]2+ was significantly less active than Rubb16, highlighting the importance of the dinuclear structure. Preliminary toxicity assays were also carried out for the ΔΔ isomers of Rubb7, Rubb10, Rubb12 and Rubb16 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 HC50 and IC50 values 100-fold higher than the MIC for the complex that showed the best potential – ΔΔ-Rubb12

Protein binding by dinuclear polypyridyl ruthenium(II) complexes and the effect of cucurbit[10]uril encapsulation

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-Rubbn; where phen = 1,10-phenanthroline, bb(n)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 ¹H 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

Tri- and tetra-nuclear polypyridyl ruthenium(II) complexes as antimicrobial agents

A series of inert tri- and tetra-nuclear polypyridylruthenium(II) complexes that are linked by the bis[4(4'- methyl-2,2'-bipyridyl)]-1,n-alkane ligand ("bbn" for n = 10, 12 and 16) have been synthesised and their potential as antimicrobial agents examined. Due to the modular nature of the synthesis of the oligonuclear complexes, it was possible to make both linear and non-linear tetra nuclear ruthenium species. The minimum inhibitory concentrations (MIC) of the ruthenium(II) complexes were determined 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). In order to gain an understanding of the relative antimicrobial activities, the cellular uptake and water–octanol partition coefficients (log P) were determined for a selection of the ruthenium complexes. Although the trinuclear complexes were the most lipophilic based upon log P values and showed the greatest cellular uptake, the linear tetranuclear complexes were generally more active, with MIC values <1 μM against the Gram positive bacteria. Similarly, although the non-linear tetranuclear complexes were slightly more lipophilic and were taken up to a greater extent by the bacteria, they were consistently less active than their linear counterparts. Of particular note, the cellular accumulation of the oligonuclear ruthenium complexes was greater in the Gram negative strains compared to that in the Gram positive S. aureus and MRSA. The results demonstrate that the lower antimicrobial activity of polypyridylruthenium(II) complexes towards Gram negative bacteria, particularly P. aeruginosa, is not strongly correlated to the cellular accumulation but rather to a lower intrinsic ability to kill the Gram negative cells

Mononuclear polypyridylruthenium(II) complexes with high membrane permeability in gram-negative bacteria—in particular Pseudomonas aeruginosa

Ruthenium(II) complexes containing the tetra dentate ligand bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane ("bb(n)"; n=10 and 12) have been synthesised and their geometric isomers separated. All [Ru(phen)(bb(n))]²⁺ (phen=1,10-phenanthroline) complexes exhibited excellent activity against Gram-positive bacteria, but only the cis-a-[Ru(phen)(bb₁₂)]²⁺ species showed good activity against Gram-negative species. In particular, the cis-a-[Ru(phen)(bb₁₂)]²⁺ complex was two to four times more active than the cis-b-[Ru(phen)(bb₁₂)]²⁺complex against the Gram-negative strains. The cis-a- and cis-b-[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-a- and cis-b-[Ru(phen)(bb₁₂)]²⁺ complexes in P. aeruginosa was considerably greater than in Escherichia coli. The uptake of the cis-a-[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-a- and cis-b-[Ru(phen)(bb(n))]²⁺ complexes exhibited relatively strong binding to DNA (Kb≈10⁶M⁻¹ ), but no significant difference between the geometricisomers was observed