Background: Targeted amplicon deep sequencing (TADS) has enabled characterization of diverse bacterial communities, yet the application of TADS to communities of parasites has been relatively slow to advance. The greatest obstacle to this has been the genetic diversity of parasitic agents, which include helminths, protozoa, arthropods, and some acanthocephalans. Meanwhile, universal amplification of conserved loci from all parasites without amplifying host DNA has proven challenging. Pan-eukaryotic PCRs preferentially amplify the more abundant host DNA, obscuring parasite-derived reads following TADS. Flaherty et al. (2018) described a pan-parasitic TADS method involving amplification of eukaryotic 18S rDNA regions possessing restriction sites only in vertebrates. Using this method, host DNA in total DNA extracts could be selectively digested prior to PCR using restriction enzymes, thereby increasing the number of parasite-derived reads obtained following NGS. This approach showed promise though was only as sensitive as conventional PCR. Results: Here, we expand on this work by designing a second set of pan-eukaryotic primers flanking the priming sites already described, enabling nested PCR amplification of the established 18S rDNA target. This nested approach facilitated introduction of a second restriction digestion between the first and second PCR, reducing the proportional mass of amplifiable host-derived DNA while increasing the number of PCR amplification cycles. We applied this method to blood specimens containing Babesia, Plasmodium, various kinetoplastids, and filarial nematodes and confirmed its limit of detection (LOD) to be approximately 10-fold lower than previously described, falling within the range of most qPCR methods. Conclusions: The assay detects and differentiates the major malaria parasites of humans, along with several other clinically important blood parasites. This represents an important step towards a TADS-based universal parasite diagnostic (UPDx) test with a sufficient LOD for routine applications.

Strongyloides stercoralis has one of the most complex life cycles of the human-infecting nematodes. A common misconception in medical and public health professions is that S. stercoralis in its biology is akin to other intestinal nematodes, such as the hookworms. Despite original evidence provided by medical and veterinary research about this unique helminth, many assumptions have entered the scientific literature. This helminth is set apart from others that commonly affect humans by (a) the internal autoinfective cycle with autoinfective larvae randomly migrating through tissue, parthenogenesis, and the potential for lifelong infection in the host, the profound pathology occurring in hyperinfection and systemic manifestations of strongyloidiasis, and (b) a limited external cycle with a single generation of free-living adults. This paper aims to review and discuss original research on the unique life cycle of S. stercoralis that distinguishes it from other helminths and highlight areas where increased understanding of the parasite’s biology might lead to improved public health prevention and control strategies.

Iron deficiency is the most common nutrient deficiency worldwide and routine supplementation is standard policy for pregnant mothers and children in most low-income countries. However, iron lies at the center of host-pathogen competition for nutritional resources and recent trials of iron administration in African and Asian children have resulted in significant excesses of serious adverse events including hospitalizations and deaths. Increased rates of malaria, respiratory infections, severe diarrhea and febrile illnesses of unknown origin have all been reported, but the mechanisms are unclear. We here investigated the ex vivo growth characteristics of exemplar sentinel bacteria in adult sera collected before and 4 h after oral supplementation with 2 mg/kg iron as ferrous sulfate. Escherichia coli, Yersinia enterocolitica and Salmonella enterica serovar Typhimurium (all gram-negative bacteria) and Staphylococcus epidermidis (gram-positive) showed markedly elevated growth in serum collected after iron supplementation. Growth rates were very strongly correlated with transferrin saturation (p < 0.0001 in all cases). Growth of Staphylococcus aureus, which preferentially scavenges heme iron, was unaffected. These data suggest that even modest oral supplements with highly soluble (non-physiological) iron, as typically used in low-income settings, could promote bacteremia by accelerating early phase bacterial growth prior to the induction of immune defenses.

The virulence factor genotypes of a large cohort of clinical, nosocomial environment and community environment isolates (184 in total) of Pseudomonas aeruginosa from Tasmania, Australia, were determined by PCR. The virulence factor genotype of the majority of isolates was highly conserved, with the exception of the virulence gene exoU, which demonstrated low prevalence (33 isolates; 18 %) in the population tested. Isolates collected from the environment of intensive therapy wards (intensive care unit and neurosurgical units) of the major tertiary referral hospital in Tasmania were found to be more likely (P<0.001 and P<0.05, respectively) to possess the virulence factor gene exoU than all other isolates. Adult cystic fibrosis isolates showed a decreased prevalence of the exoU gene (P<0.01) when compared to other clinical isolates (P<0.01), which may indicate decreased virulence. No specific virulence factor genotype was associated with the cystic fibrosis epidemic strains tested.

SUMMARY: Strongyloidiasis is a World Health Organization neglected tropical disease usually caused by Strongyloides stercoralis , a parasitic worm with a complex life cycle. Globally, 300–600 million people are infected through contact with fecally contaminated soil. An autoinfective component of the life cycle can lead to chronic infection that may be asymptomatic or cause long-term symptoms, including malnourishment in children. Low larval output can limit the sensitivity of detection in stool, with serology being effective but less sensitive in immunocompromise. Host immunosuppression can trigger catastrophic, fatal hyperinfection/dissemination, where large numbers of larvae pierce the bowel wall and disseminate throughout the organs. Stable disease is effectively treated by single-dose ivermectin, with disease in immunocompromised patients treated with multiple doses. Strategies for management include raising awareness, clarifying zoonotic potential, the development and use of effective diagnostic tests for epidemiological studies and individual diagnosis, and the implementation of treatment programs with research into therapeutic alternatives and medication safety.

Advances in laboratory techniques have revolutionized parasitology diagnostics over the past several decades. Widespread implementation of rapid antigen detection tests has greatly expanded access to tests for global parasitic threats such as malaria, while next-generation amplification and sequencing methods allow for sensitive and specific detection of human and animal parasites in complex specimen matrices.