Andrew R. Greenhill

Strongyloidiasis, caused by nematodes of the genus Strongyloides , is a neglected tropical disease with significant public health implications. Accurate diagnostics are essential for disease surveillance and control, yet traditional stool-based and serological methods have limitations in sensitivity and specificity. Molecular assays offer an alternative diagnostic approach. In this study, we compared four probe-based real-time PCR (qPCR) assays and one loop-mediated isothermal amplification (LAMP) assay using human fecal DNA samples from Gabon and Papua New Guinea with both Strongyloides stercoralis and Strongyloides fuelleborni infections. Our results revealed discordance between assays, with no single primer set identifying all positive samples across all assays. One assay was identified as specific for S. stercoralis only, while other assays amplify other Strongyloides spp. This may impact the specificity of molecular diagnostics in epidemiological surveys, particularly with increasing identifications of S. fuelleborni infections in humans from Asia and New Guinea. Of the assays examined, four were probe-based qPCR assays and a LAMP assay. On sequencing positive amplicons, enteric bacterial sequences were identified for two assays. However, subsequent testing and BLAST results identified that including a probe, as the assays were designed, would increase specificity, so that the bacterial sequences would not be amplified. These findings provide a context for the interpretation of results from various Strongyloides molecular assays. IMPORTANCE: Strongyloidiasis is an underdiagnosed neglected parasitic infection that poses a growing challenge for public health surveillance and control, particularly in regions where multiple Strongyloides species exist and clinical case detection is challenging. Molecular assays are increasingly used as reference diagnostic tools, yet their performance and species specificity have not been systematically evaluated across diverse endemic settings. In this study, we tested the hypothesis that commonly used molecular assays differ in their ability to accurately and consistently detect and discriminate Strongyloides stercoralis and Strongyloides fuelleborni in human fecal samples. By comparing five molecular assays using samples from Gabon and Papua New Guinea, we demonstrate discordance between tests, including species-dependent detection biases. These findings provide evidence that molecular diagnostics may misrepresent species distribution. Our results highlight the necessity for awareness of target species specificity and for regionally validated molecular tools to ensure the reliable interpretation of assay results.

Background: Respiratory infections are the leading cause of childhood morbidity and mortality in Papua New Guinea (PNG). Evidence suggests probiotics may reduce early‑life infections. We evaluated the safety and feasibility of two probiotics in PNG newborns and explored associations with gut detection, infections, vaccine responses, and nasopharyngeal bacterial carriage.Methods: We conducted a randomised placebo‑controlled double-blinded trial, assigning healthy newborns (1:1:1) within 72 hours from birth to 7‑day supplementation with Lactiplantibacillus plantarum+fructo‑oligosaccharide, Bifidobacterium infantis+2′‑fucosyllactose, or placebo.Findings: From 29-October-2020 to 30-June-2023, 244 infants were enrolled. 89% completed 7 doses. Both probiotics were safe and well-tolerated, with comparable adverse events rates across groups. Probiotic supplementation was not associated with increased or decreased infection risk. B. infantis was detected in 13.1% of all newborns on Day 1, was highest in supplemented infants on Day 8 (B. infantis, 89.5%; L. plantarum, 50.7%; placebo, 44.3%; p<0.001) and comparable between groups afterwards. L. plantarum detection was highest after supplementation on Day 8 (L. plantarum, 45.3%; B. infantis, 4.0%; placebo, 2.5%; p<0.001) and declined to <3% in any group from 1 month. Carriage of S. pneumoniae (30.3% at 2 weeks, 79.4% at 4 months), H. influenzae (18.1% at 2 weeks, 54.3% at 4 months), and S. aureus (53.4% at 2 weeks, 15.4% at 4 months) was comparable between groups. Vaccine responses were also comparable. Interpretation: Probiotic supplementation was feasible and safe. B. infantis was prevalent in PNG newborns regardless of supplementation. L. plantarum supplementation was associated with transient gut detection.

Background: Childhood mortality in low- or middle-income countries (LMICs) remains a major public health concern, with infections being a leading cause of infant death. Probiotics have shown promise in reducing infection-related morbidity and mortality in preterm infants, but their use in newborns born at or near term in LMICs requires further investigation. Objective: This study aims to assess the feasibility, safety, and initial outcomes of administering 1 of 2 synbiotic formulations or a placebo to newborns in the Eastern Highlands Province of Papua New Guinea. Methods: Following ethics approvals in 2018 and 2019, and receipt of the synbiotic preparations in September 2020, healthy neonates (<72 h old; n=244) were recruited between October 2020 and June 2023 and randomly assigned in a double-blind manner (1:1:1) to receive an oral preparation containing Lactiplantibacillus plantarum, Bifidobacterium longum subspecies infantis, or placebo for 7 consecutive days. Follow-up continued for 6 months, with rectal swabs, stool, blood, saliva, and nasopharyngeal swabs collected before the intervention; on day 7; at age 2 weeks; and at ages 1, 3, 4, and 6 months. Ongoing analyses will assess probiotic gut colonization, bacterial nasopharyngeal carriage, and antibody responses to routine childhood vaccines (Hemophilus influenzae type b; hepatitis B; 13-valent pneumococcal conjugate vaccine; and diphtheria, tetanus, and whole-cell pertussis) as well as hospitalization and infection rates among the intervention groups compared to the placebo group. Results: Recruitment began in October 2020, with the target sample size expanded from 195 to 240 in March 2023 owing to higher-than-anticipated loss to follow-up during the COVID-19 pandemic. Of the 244 enrolled infants, 218 (89.3%) completed the full 7-day synbiotic or placebo course, and 169 (69.3%) completed the study. All follow-up visits concluded in December 2023. Disruptions due to the COVID-19 pandemic led to family relocations outside the study area, preventing some infants (75/244, 30.7%) from completing the study. High rates of sample collection were achieved, with rectal swabs (1452/1474, 98.1%), nasopharyngeal swabs (1255/1262, 99.44%), saliva samples (881/882, 99.9%), and blood samples (876/882, 99.3%) successfully obtained at multiple time points. Data analysis is ongoing and expected to be completed by the end of 2025. Conclusions: This study demonstrates that probiotic supplementation is feasible and safe in healthy infants in an LMIC such as Papua New Guinea. The findings from this study will inform the design of larger trials of probiotics and synbiotics to complement existing efforts to reduce infection-related infant mortality in LMICs, while also providing insights into their clinical, immunological, and microbiological impacts in infants. Trial Registration: Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12620001369910; https://anzctr.org.au/Trial/Registration/TrialReview.aspx?ACTRN=12620001369910 International Registered Report Identifier (IRRID): DERR1-10.2196/73926

The island of New Guinea is known to harbour a unique human infecting species of Strongyloides : Strongyloides fuelleborni subsp. kellyi . Stool DNA extracts (n=164) from 19 PNG infants, collected over 13 months, were analysed using Strongyloides real-time PCRs and underwent metabarcoding of cox1 , 18S rRNA HVR-I and HVR-IV loci. Eight infants were infected with Strongyloides ; seven identified as Strongyloides fuelleborni fuelleborni and one as S. f. kellyi . Phylogenetic and haplotyping analyses indicated that S. f. fuelleborni in PNG belongs to the Indochina sub-clade of S. f. fuelleborni, and S. f. kellyi does not represent a subspecies of S. fuelleborni . We provide the first molecular evidence of S. f. fuelleborni infection in humans in the Pacific. We also support that S. f. kellyi is a separate and distinct species of human infecting helminth. Renewed clinical and epidemiological investigations into infant strongyloidiasis in New Guinea using species-discriminatory molecular tools are indicated.

BACKGROUND Childhood mortality in low- or middle-income countries (LMICs) remains a major public health concern, with infections being a leading cause of infant death. Probiotics have shown promise in reducing infection-related morbidity and mortality in preterm infants, but their use in newborns born at or near term in LMICs requires further investigation. OBJECTIVE This study aims to assess the feasibility, safety, and initial outcomes of administering 1 of 2 synbiotic formulations or a placebo to newborns in the Eastern Highlands Province of Papua New Guinea. METHODS Following ethics approvals in 2018 and 2019, and receipt of the synbiotic preparations in September 2020, healthy neonates (<72 h old; n=244) were recruited between October 2020 and June 2023 and randomly assigned in a double-blind manner (1:1:1) to receive an oral preparation containing Lactiplantibacillus plantarum, Bifidobacterium longum subspecies infantis, or placebo for 7 consecutive days. Follow-up continued for 6 months, with rectal swabs, stool, blood, saliva, and nasopharyngeal swabs collected before the intervention; on day 7; at age 2 weeks; and at ages 1, 3, 4, and 6 months. Ongoing analyses will assess probiotic gut colonization, bacterial nasopharyngeal carriage, and antibody responses to routine childhood vaccines (Hemophilus influenzae type b; hepatitis B; 13-valent pneumococcal conjugate vaccine; and diphtheria, tetanus, and whole-cell pertussis) as well as hospitalization and infection rates among the intervention groups compared to the placebo group. RESULTS Recruitment began in October 2020, with the target sample size expanded from 195 to 240 in March 2023 owing to higher-than-anticipated loss to follow-up during the COVID-19 pandemic. Of the 244 enrolled infants, 218 (89.3%) completed the full 7-day synbiotic or placebo course, and 169 (69.3%) completed the study. All follow-up visits concluded in December 2023. Disruptions due to the COVID-19 pandemic led to family relocations outside the study area, preventing some infants (75/244, 30.7%) from completing the study. High rates of sample collection were achieved, with rectal swabs (1452/1474, 98.1%), nasopharyngeal swabs (1255/1262, 99.44%), saliva samples (881/882, 99.9%), and blood samples (876/882, 99.3%) successfully obtained at multiple time points. Data analysis is ongoing and expected to be completed by the end of 2025. CONCLUSIONS This study demonstrates that probiotic supplementation is feasible and safe in healthy infants in an LMIC such as Papua New Guinea. The findings from this study will inform the design of larger trials of probiotics and synbiotics to complement existing efforts to reduce infection-related infant mortality in LMICs, while also providing insights into their clinical, immunological, and microbiological impacts in infants. CLINICALTRIAL Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12620001369910; https://anzctr.org.au/Trial/Registration/TrialReview.aspx?ACTRN=12620001369910 INTERNATIONAL REGISTERED REPORT DERR1-10.2196/73926

Antibiotic resistance is a global health crisis. Roughly two-thirds of all antibiotics used are in production animals, which has the potential to impact on the development of antibiotic resistance in bacterial pathogens of humans. There is little visibility on the extent of antibiotic resistance in the Australian food chain. This study sought to establish the incidence of antibiotic resistance among enterococci from poultry in Victoria. In 2016, poultry from a Victorian processing facility were swabbed immediately post-slaughter and cultured for Enterococcus species. All isolates recovered were speciated and tested for antibiotic susceptibility to twelve antibiotics following the Clinical Laboratory Standards Institute guidelines. Six farms and 207 birds were sampled and from these 285 isolates of Enterococcus were recovered. Eight different enterococcal species were identified: E. faecalis (n = 122; 43%), E. faecium (n = 92; 32%), E. durans (n = 35; 12%), E. thailandicus (n = 23; 8%), E. hirae (n = 10; 3%) and a single each of E. avium, E. gallinarum, and E. mundtii. Reduced susceptibility to older classes of antibiotic were common, in particular: erythromycin (73%), rifampin (49%), nitrofurantoin (40%) and ciprofloxacin (39%). Two vancomycin intermediate isolates were recovered, but no resistance was detected to either linezolid or gentamicin. The relatively high numbers of a recently described species, E. thailandicus, suggests this species might be well adapted to colonise poultry. The incidence of antibiotic resistance is lower in isolates from poultry than in human medicine in Australia. These results suggest that poultry may serve as a reservoir for older antibiotic resistance genes but is not driving the emergence of antimicrobial resistance in human bacterial pathogens. This is supported by the absence of resistance to linezolid and gentamicin.

The Global Typhoid Genomics Consortium was established to bring together the typhoid research community to aggregate and analyse Salmonella enterica serovar Typhi (Typhi) genomic data to inform public health action. This analysis, which marks twenty-one years since the publication of the first Typhi genome, represents the largest Typhi genome sequence collection to date (n=13,000), and provides a detailed overview of global genotype and antimicrobial resistance (AMR) distribution and temporal trends, generated using open analysis platforms (GenoTyphi and Pathogenwatch). Compared with previous global snapshots, the data highlight that genotype 4.3.1 (H58) has not spread beyond Asia and Eastern/Southern Africa; in other regions, distinct genotypes dominate and have independently evolved AMR. Data gaps remain in many parts of the world, and we show potential of travel-associated data to provide informal “sentinel” surveillance for such locations. The data indicate ciprofloxacin non-susceptibility (>1 resistance determinant) is widespread across geographies and genotypes, with high-level resistance (≥3 determinants) reaching 20% prevalence in South Asia. Extensively drug-resistant (XDR) typhoid has become dominant in Pakistan (70% in 2020), but has not yet become established elsewhere. Ceftriaxone resistance has emerged in eight non-XDR genotypes, including a ciprofloxacin-resistant lineage (4.3.1.2.1) in India. Azithromycin resistance mutations were detected at low prevalence in South Asia, including in two common ciprofloxacin-resistant genotypes. The Consortium’s aim is to encourage continued data sharing and collaboration to monitor the emergence and global spread of AMR Typhi, and to inform decision-making around the introduction of typhoid conjugate vaccines (TCVs) and other prevention and control strategies.

Antibiotic resistance is an ongoing threat to both human and animal health. Migratory birds are a potential vector for the spread of novel pathogens and antibiotic resistance genes. To date, there has been no comprehensive study investigating the presence of antibiotic resistance (AMR) in the bacteria of Australian shorebirds or terns. In the current study, 1022 individual birds representing 12 species were sampled across three states of Australia (Victoria, South Australia, and Western Australia) and tested for the presence of phenotypically resistant strains of three bacteria with potential to be zoonotic pathogens; Escherichia coli, Enterococcusspp., and Salmonellasp. In total, 206 E. coli, 266 Enterococcusspp., and 20 Salmonellasp. isolates were recovered, with AMR detected in 42% of E. coli, 85% of Enterococcusspp., and 10% of Salmonellasp. Phenotypic resistance was commonly detected to erythromycin (79% of Enterococcusspp.), ciprofloxacin (31% of Enterococcusspp.) and streptomycin (21% of E. coli). Resident birds were more likely to carry AMR bacteria than migratory birds (p ≤ .001). Bacteria isolated from shorebirds and terns are commonly resistant to at least one antibiotic, suggesting that wild bird populations serve as a potential reservoir and vector for AMR bacteria. However, globally emerging phenotypes of multidrug‐resistant bacteria were not detected in Australian shorebirds. This study provides baseline data of the carriage of AMR bacteria in Australian shorebirds and terns.