The molecular characterisation of shigella spp. from Papua New Guinea and Pacific Island Nations

In Papua New Guinea (PNG) and other low-middle income countries (LMICs) in Oceania, diarrhoea remains a leading cause of hospitalisation and death in children <5 years old; and is an important cause of illness in older children and adults. Shigella is one of four leading causes of diarrhoea globally and is likely a major cause of diarrhoea in Oceanic LMICs, particularly PNG. A survey was conducted among parents of children with diarrhoea in the PNG highlands, demonstrating limited access to, and application of, sanitation and hygiene; likely contributing to the transmission of Shigella and other pathogens in PNG. To better understand Shigella in Oceanic LMICs, multiple analyses of isolates from within PNG (n=38), far-northeastern Australia, and from travellers returning to Australia from Oceanic LMICs with shigellosis (n=34) were conducted. Antimicrobial susceptibility (by disc diffusion), detection of virulence genes by polymerase chain reaction (PCR) were conducted on all isolates, and whole genome sequencing (WGS) conducted on 63 isolates. Shigella spp. were commonly resistant to two or more classes of first-line antibiotics, with resistance more common in post-2010 relative to pre-2010 isolates. WGS was used to verify PCR detection of virulence genes, determine whether resistance could be predicted genetically, and conduct phylogenetic analysis of Shigella spp. in Oceania. WGS surpassed PCR in the detection of virulence genes, but correlated poorly with phenotypic antimicrobial resistance. Phylogenetic analysis revealed the intra- and inter-country relatedness. Three phylogenetic groups of S. flexneri co-exist in Oceanic LMICs (and far-northeastern Australia), the result of multiple incursions. Two lineages of S. sonnei were detected, one circulating in PNG and New Caledonia, and the other in various other countries including Fiji; with no geographical overlap of the two S. sonnei lineages. Incursions of Shigella into the Oceanic LMICs occur regularly, and are likely to occur again. This study provides evidence of the need for, and potential approach to, expanded surveillance of Shigella in the region.Doctor of Philsoph

Significant geographical differences in prevalence of mutations associated with Plasmodium falciparum and Plasmodium vivax drug resistance in two regions from Papua New Guinea

Drug resistance remains a major obstacle to malaria treatment and control. It can arise and spread rapidly, and vary substantially even at sub-national level. National malaria programmes require cost-effective and timely ways of characterizing drug-resistance at multiple sites within their countries.; An improved multiplexed post-PCR ligase detection reaction-fluorescent microsphere assay (LDR-FMA) was used to simultaneously determine the presence of mutations in chloroquine resistance transporter (crt), multidrug resistance 1 (mdr1), dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes in Plasmodium falciparum (n = 727) and Plasmodium vivax (n = 574) isolates collected in 2006 from cross-sectional community population surveys in two geographically distinct regions (Madang and East Sepik) of Papua New Guinea (PNG) where strong regional differences in in vivo aminoquinoline and antifolate therapeutic efficacy had previously been observed. Data were compared to those of a follow-up survey conducted in 2010.; Despite some very low parasite densities, the assay successfully amplified all P. falciparum and P. vivax loci in 77 and 69 % of samples, respectively. In 2006, prevalences of pfdhfr (59R-108 N) double mutation/wild type pfdhps haplotype, pfcrt SVMNT haplotype (72S-76T double mutation), and 86Y pfmdr1 mutation all exceeded 90 %. For P. vivax, 65 % carried at least two pvdhfr mutations, 97 % the 647P pvdhps mutation and 54 % the 976F pvmdr1 mutation. Prevalence of mutant haplotypes was higher in Madang than East Sepik for pfcrt SVMNT (97.4 vs 83.3 %, p = 0.001), pfdhfr (59R-108 N) (100 vs 90.6 %, p = 0.001), pvdhfr haplotypes (75.8 vs 47.6 %, p = 0.001) and pvmdr1 976F (71.2 vs 26.2 %, p &lt; 0.001). Data from a subsequent Madang survey in 2010 showed that the prevalence of pfdhps mutations increased significantly from &lt;5 % to &gt;30 % (p &lt; 0.001) as did the prevalence of pvdhfr mutant haplotypes (from 75.8 to 97.4 %, p = 0.012).; This LDR-FMA multiplex platform shows feasibility for low-cost, high-throughput, rapid characterization of a broad range of drug-resistance markers in low parasitaemia infections. Significant geographical differences in mutation prevalence correlate with previous genotyping surveys and in vivo trials and may reflect variable drug pressure and differences in health-care access in these two PNG populations

Shigellosis : A truly neglected disease in Papua New Guinea

Diarrhoeal diseases still affect many people, especially children living in impoverished and under-developed settings. In Papua New Guinea (PNG) diarrhoea remains one of the leading causes of hospitalization and a major cause of death. Here, we focus on the role of Shigella in diarrhoeal illness in PNG, and provide an overview of the causative organism and the illness. A review of the available data on the aetiology of diarrhoea in PNG suggests that shigellosis is a major cause of diarrhoeal illness. Since shigellosis can cause protracted and life-threatening illness an appreciation of the burden of shigellosis is important to aid in the development of optimal prevention and control strategies. Treatment strategies for all cases of moderate-severe diarrhoeal illness should centre on rehydration, but where antimicrobial treatment is required consideration should be given to the increasing antimicrobial resistance observed in Shigella isolates in PNG

Shigellosis: a truly neglected disease in Papua New Guinea

Diarrhoeal diseases still affect many people, especially children living in impoverished and under-developed settings. In Papua New Guinea (PNG) diarrhoea remains one of the leading causes of hospitalization and a major cause of death. Here, we focus on the role of Shigella in diarrhoeal illness in PNG, and provide an overview of the causative organism and the illness. A review of the available data on the aetiology of diarrhoea in PNG suggests that shigellosis is a major cause of diarrhoeal illness. Since shigellosis can cause protracted and life-threatening illness an appreciation of the burden of shigellosis is important to aid in the development of optimal prevention and control strategies. Treatment strategies for all cases of moderate-severe diarrhoeal illness should centre on rehydration, but where antimicrobial treatment is required consideration should be given to the increasing antimicrobial resistance observed in Shigella isolates in PNG

Antimicrobial sensitivity trends and virulence genes in Shigella spp. from the Oceania region

Shigella is a common cause of diarrhoea in Papua New Guinea (PNG) and other Oceania countries. However, little is known about the strains causing infection. Archived Shigella isolates (n = 72) were obtained from research laboratories in PNG and reference laboratories in Australia. Shigella virulence genes were detected by PCR, and antimicrobial susceptibility was determined by disk diffusion. The ipaH virulence gene was present in all 72 isolates. The prevalence of other virulence genes was variable, with ial, invE, ipaBCD, sen/ospD3 and virF present in 60% of isolates and set1A and set1B genes present in 42% of isolates. Most S. flexneri isolates contained genes encoding enterotoxin 1 and/or enterotoxin 2. Resistance to antibiotics was common, with 51/72 isolates resistant to 2-4 antimicrobials. A greater proportion of bacteria isolated since 2010 (relative to pre-2010 isolates) were resistant to commonly used antibiotics such as ampicillin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole; suggesting that antimicrobial resistance (AMR) in Shigella is increasing over time in the Oceania region. There is a need for improved knowledge regarding Shigella circulation in the Oceania region and further monitoring of AMR patterns

Plasmodium falciparum and Plasmodium vivax genotypes and efficacy of intermittent preventive treatment in Papua New Guinea

Intermittent preventive treatment of infants (IPTi) reduces early childhood malaria-related morbidity. While genotypic drug resistance markers have proven useful in predicting the efficacy of antimalarial drugs in case management, there are few equivalent data relating to their protective efficacy when used as IPTi. The present data from an IPTi trial in Papua New Guinea demonstrate how these markers can predict protective efficacy of IPTi for both Plasmodium falciparum and Plasmodium vivax

Outgroups used for phylogenetic tree rooting

Table includes alleles from representative non-genotype 2.1.7 sequences as outgroups for phylogenetic tree rooting

Excluded repeat and phage regions in PNG MDUST348 2.1.7.2 completed reference sequence

This table details regions filtered from PNG genotype 2.1.7 alignment, where repetitive regions were identified using the ncumer command in MUMmer (v3.23) and integrated prophage sequences detected using the PHAge Search Tool Enhanced Release. Further recombination were removed with Gubbins (v2.3.2)

Accession numbers and data for S. Typhi sequences used in this study

Details of antimicrobial susceptibility testing (AST) for n = 40 (88.9%) of travel-associated cases for chloramphenicol, ampicillin, sulfamethoxazole, trimethoprim, nalidixic acid, tetracycline, kanamycin, gentamycin, and spectinomycin. This includes the individual accession numbers for nucleotide sequences