Andrew R. Greenhill

The Gut Microbiota of Rural Papua New Guineans: Composition, Diversity Patterns, and Ecological Processes

Although recent research revealed an impact of westernization on diversity and composition of the human gut microbiota, the exact consequences on metacommunity characteristics are insufficiently understood, and the underlying ecological mechanisms have not been elucidated. Here, we have compared the fecal microbiota of adults from two non-industrialized regions in Papua New Guinea (PNG) with that of United States (US) residents. Papua New Guineans harbor communities with greater bacterial diversity, lower inter-individual variation, vastly different abundance profiles, and bacterial lineages undetectable in US residents. A quantification of the ecological processes that govern community assembly identified bacterial dispersal as the dominant process that shapes the microbiome in PNG but not in the US. These findings suggest that the microbiome alterations detected in industrialized societies might arise from modern lifestyle factors limiting bacterial dispersal, which has implications for human health and the development of strategies aimed to redress the impact of westernization.

Evaluation of Serological Diagnostic Tests for Typhoid Fever in Papua New Guinea Using a Composite Reference Standard

ABSTRACT Typhoid fever remains a major global health problem. A major impediment to improving outcomes is the lack of appropriate diagnostic tools, which have not significantly improved in low-income settings for 100 years. We evaluated two commercially available rapid diagnostic tests (Tubex and TyphiDot), a prototype (TyphiRapid TR-02), and the commonly used single-serum Widal test in a previously reported high-burden area of Papua New Guinea. Samples were collected from 530 outpatients with axillary temperatures of ≥37.5°C, and analysis was conducted on all malaria-negative samples (n = 500). A composite reference standard of blood culture and PCR was used, by which 47 participants (9.4%) were considered typhoid fever positive. The sensitivity and specificity of the Tubex (51.1% and 88.3%, respectively) and TyphiDot (70.0% and 80.1%, respectively) tests were not high enough to warrant their ongoing use in this setting; however, the sensitivity and specificity for the TR-02 prototype were promising (89.4% and 85.0%, respectively). An axillary temperature of ≥38.5°C correlated with typhoid fever (P = 0.014). With an appropriate diagnostic test, conducting typhoid fever diagnosis only on patients with high-grade fever could dramatically decrease the costs associated with diagnosis while having no detrimental impact on the ability to accurately diagnose the illness.

Groundwater Seeps Facilitate Exposure to Burkholderia pseudomallei

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 samples (n = 40) and 92.5% (95% CI, 83.9 to 100) of seasonal groundwater samples (n = 40). Further sampling 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 samples (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 samples 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.

Clonal Origins of Vibrio cholerae O1 El Tor Strains, Papua New Guinea, 2009-2011

We used multilocus sequence typing and variable number tandem repeat analysis to determine the clonal origins of Vibrio cholerae O1 El Tor strains from an outbreak of cholera that began in 2009 in Papua New Guinea. The epidemic is ongoing, and transmission risk is elevated within the Pacific region.

Multilocus Sequence Typing of Streptococcus pneumoniae by Use of Mass Spectrometry

ABSTRACT Multilocus sequence typing (MLST) is an important tool for the global surveillance of bacterial pathogens that is performed by comparing the sequences of designated housekeeping genes. We developed and tested a novel mass spectrometry-based method for MLST of Streptococcus pneumoniae. PCR amplicons were subjected to in vitro transcription and base-specific cleavage, followed by analysis of the resultant fragments by using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Comparison of the cleavage fragment peak patterns to a reference sequence set permitted automated identification of alleles. Validation experiments using 29 isolates of S. pneumoniae revealed that the results of MALDI-TOF MS MLST matched those obtained by traditional sequence-based MLST for 99% of alleles and that the MALDI-TOF MS method accurately identified two single-nucleotide variations. The MADLI-TOF MS method was then used for MLST analysis of 43 S. pneumoniae isolates from Papua New Guinean children. The majority of the isolates present in this population were not clonal and contained seven new alleles and 30 previously unreported sequence types.

Respiratory viral pathogens associated with lower respiratory tract disease among young children in the highlands of Papua New Guinea.

Abstract Background Acute lower respiratory tract infections (ALRI) commonly result in fatal outcomes in the young children of Papua New Guinea (PNG). However, comprehensive studies of the viral aetiology of ALRI have not been conducted in PNG for almost 30 years. Objectives To determine the viruses associated with ALRI among children living in the PNG highlands using sensitive molecular detection techniques. Study design Pernasal swabs were collected routinely between 1 week and 18 months of age and also during episodes of ALRI, as part of a neonatal pneumococcal conjugate vaccine trial. A tandem multiplex real-time PCR assay was used to test for a comprehensive range of respiratory viruses in samples collected from 221 young children. Picornavirus typing was supported by DNA sequence analysis. Results Recognized pathogenic respiratory viruses were detected in 198/273 (73%) samples collected from children with no evidence of ALRI and 69/80 (86%) samples collected during ALRI episodes. Human rhinoviruses (HRV) species A, B and C were detected in 152 (56%) samples from non-ALRI children and 50 (63%) samples collected during ALRI episodes. Partial structural region sequences for two new species C rhinoviruses were added to the GenBank database. ALRI was associated with detection of adenovirus species B (p <0.01) or C (p <0.05), influenza A (p <0.0001) or respiratory syncytial virus (p <0.0001). Multiple viruses were detected more often during ALRI episodes (49%) than when children displayed no symptoms of ALRI (18%) (p <0.0001). Conclusions The burden of infection with respiratory viruses remains significant in young children living in the PNG highlands.

Molecular phylogeny of Burkholderia pseudomallei from a remote region of Papua New Guinea.

Background The island of New Guinea is located midway between the worlds two major melioidosis endemic regions of Australia and Southeast Asia. Previous studies in Papua New Guinea have demonstrated autochthonous melioidosis in Balimo, Western province. In contrast to other regions of endemicity, isolates recovered from both environmental and clinical sources demonstrate narrow genetic diversity over large spatial and temporal scales. Methodology/Principal Findings We employed molecular typing techniques to determine the phylogenetic relationships of these isolates to each other and to others worldwide to aid in understanding the origins of the Papua New Guinean isolates. Multi-locus sequence typing of the 39 isolates resolved three unique sequence types. Phylogenetic reconstruction and Structure analysis determined that all isolates were genetically closer to those from Australia than those from Southeast Asia. Gene cluster analysis however, identified a Yersinia-like fimbrial gene cluster predominantly found among Burkholderia pseudomallei derived from Southeast Asia. Higher resolution VNTR typing and phylogenetic reconstruction of the Balimo isolates resolved 24 genotypes with long branch lengths. These findings are congruent with long term persistence in the region and a high level of environmental stability. Conclusions/Significance Given that anthropogenic influence has been hypothesized as a mechanism for the dispersal of B. pseudomallei, these findings correlate with limited movement of the indigenous people in the region. The palaeogeographical and anthropogenic history of Australasia and the results from this study indicate that New Guinea is an important region for the further study of B. pseudomallei origins and dissemination.

Evaluation of colorimetric detection methods for Shigella, Salmonella, and Vibrio cholerae by loop-mediated isothermal amplification

We evaluated loop-mediated isothermal amplification end-point detection methods for Salmonella, Shigella, and Vibrio cholerae. Detection sensitivities were comparable to real-time PCR methods. The colorimetric dyes hydroxynaphthol blue and SYBR Green I showed increased sensitivity when compared to visual and automated turbidity readings. End-point colorimetric dyes promise great utility in developing settings.

Detection of enteric viral and bacterial pathogens associated with paediatric diarrhoea in Goroka, Papua New Guinea

OBJECTIVES The aim of this study was to investigate the viral and bacterial causes of acute watery diarrhoea in hospitalized children in Papua New Guinea. METHODS A retrospective analysis was conducted on stool samples collected from 199 children (age <5 years) admitted to the paediatric ward of Goroka General Hospital from August 2009 through November 2010. A large range of viral and bacterial enteric pathogens were targeted using real-time PCR/RT-PCR assays. RESULTS Young children were much more likely to be admitted with acute gastroenteritis, with 62.8% of patients aged <1 year and 88.4% aged <2 years. An enteric pathogen was detected in 69.8% (n=138) of patients. The most commonly detected pathogens were Shigella spp (26.6%), rotavirus (25.6%), adenovirus types 40/41 (11.6%), enterotoxigenic Escherichia coli (11.1%), enteropathogenic E. coli (8.5%), norovirus G2 (6.0%), and Campylobacter spp (4.0%). Norovirus G1, sapovirus, and Salmonella spp were also detected, but below our statistical limit of detection. Vibrio cholerae and astrovirus were not detected in any patients. Mixed infections were detected in 22.1% of patients, with Shigella and rotavirus most commonly detected in co-infections with other pathogens. CONCLUSIONS This study demonstrates that Shigella and rotavirus are the major pathogens associated with acute paediatric gastroenteritis in this setting.

Susceptibility of primary human endothelial cells to C. perfringens beta-toxin suggesting similar pathogenesis in human and porcine necrotizing enteritis.

Clostridium perfringens type C causes fatal necrotizing enteritis in different mammalian hosts, most commonly in newborn piglets. Human cases are rare, but the disease, also called pigbel, was endemic in the Highlands of Papua New Guinea. Lesions in piglets and humans are very similar and characterized by segmental necro-hemorrhagic enteritis in acute cases and fibrino-necrotizing enteritis in subacute cases. Histologically, deep mucosal necrosis accompanied by vascular thrombosis and necrosis was consistently reported in naturally affected pigs and humans. This suggests common pathogenetic mechanisms. Previous in vitro studies using primary porcine aortic endothelial cells suggested that beta-toxin (CPB) induced endothelial damage contributes to the pathogenesis of C. perfringens type C enteritis in pigs. In the present study we investigated toxic effects of CPB on cultured primary human macro- and microvascular endothelial cells. In vitro, these cells were highly sensitive to CPB and reacted with similar cytopathic and cytotoxic effects as porcine endothelial cells. Our results indicate that porcine and human cell culture based in vitro models represent valuable tools to investigate the pathogenesis of this bacterial disease in animals and humans.