Parasitic nematodes infect over 1 billion people worldwide and cause some of the most common neglected tropical diseases. Despite their prevalence, our understanding of the biology of parasitic nematodes has been limited by the lack of tools for genetic intervention. In particular, it has not yet been possible to generate targeted gene disruptions and mutant phenotypes in any parasitic nematode. Here, we report the development of a method for introducing CRISPR-Cas9-mediated gene disruptions in the human-parasitic threadworm Strongyloides stercoralis. We disrupted the S. stercoralis twitchin gene unc-22, resulting in nematodes with severe motility defects. Ss-unc-22 mutations were resolved by homology-directed repair when a repair template was provided. Omission of a repair template resulted in deletions at the target locus. Ss-unc-22 mutations were heritable; we passed Ss-unc-22 mutants through a host and successfully recovered mutant progeny. Using a similar approach, we also disrupted the unc-22 gene of the rat-parasitic nematode Strongyloides ratti. Our results demonstrate the applicability of CRISPR-Cas9 to parasitic nematodes, and thereby enable future studies of gene function in these medically relevant but previously genetically intractable parasites.

Skin-penetrating parasitic nematodes infect approximately one billion people worldwide and are a major source of neglected tropical disease [1-6]. Their life cycle includes an infective third-larval (iL3) stage that searches for hosts to infect in a poorly understood process that involves both thermal and olfactory cues. Here, we investigate the temperature-driven behaviors of skin-penetrating iL3s, including the human-parasitic threadworm Strongyloides stercoralis and the human-parasitic hookworm Ancylostoma ceylanicum. We show that human-parasitic iL3s respond robustly to thermal gradients. Like the free-living nematode Caenorhabditis elegans, human-parasitic iL3s show both positive and negative thermotaxis, and the switch between them is regulated by recent cultivation temperature [7]. When engaging in positive thermotaxis, iL3s migrate toward temperatures approximating mammalian body temperature. Exposing iL3s to a new cultivation temperature alters the thermal switch point between positive and negative thermotaxis within hours, similar to the timescale of thermal plasticity in C. elegans [7]. Thermal plasticity in iL3s may enable them to optimize host finding on a diurnal temperature cycle. We show that temperature-driven responses can be dominant in multisensory contexts such that, when thermal drive is strong, iL3s preferentially engage in temperature-driven behaviors despite the presence of an attractive host odorant. Finally, targeted mutagenesis of the S. stercoralis tax-4 homolog abolishes heat seeking, providing the first evidence that parasitic host-seeking behaviors are generated through an adaptation of sensory cascades that drive environmental navigation in C. elegans [7-10]. Together, our results provide insight into the behavioral strategies and molecular mechanisms that allow skin-penetrating nematodes to target humans.

Purpose:Infectious keratitis is a vision-threatening condition requiring close follow-up and disciplined eye drop administration to achieve resolution. Although patients presenting to county hospitals often have more severe presentations, there is a paucity of risk and outcomes data in this setting. This study investigates risk factors predicting loss to follow-up (LTFU), medication noncompliance, and poor outcomes for infectious keratitis in the county hospital setting.Methods:This was a retrospective case-control study at Zuckerberg San Francisco General Hospital and Trauma Center. Inclusion criteria were patients who had corneal cultures for suspected infectious bacterial or fungal keratitis between 2010 and 2021. Exclusion criteria were patients with viral keratitis only. Multivariable logistic regression was used to analyze the relationship of social and medical risk factors with LTFU, medication noncompliance, worsened visual acuity (VA), and delayed resolution time.Results:Of 174 patients with infectious keratitis in this analysis, 69 (40.0%) had LTFU. Unemployment was associated with increased risk of LTFU (odds ratio 2.58, P = 0.049) and worse final VA (P = 0.001). Noncompliance trended toward an association with homelessness (odds ratio 3.48, P = 0.095). Increasing age correlated with longer resolution time, with each 1-year increase associated with delayed resolution by 0.549 days (P = 0.042).Conclusions:Patients experiencing unemployment, homelessness, or increased age demonstrate higher risk for treatment barriers including loss to follow-up and medication noncompliance, resulting in worse VA and delayed time to resolution. These risk factors should be considered when determining the need for more deliberate follow-up measures in patients with infectious keratitis.

Purpose: The aim of this study was to determine risk factors and clinical course of corneal ulcers in the setting of opioid use. Methods: A retrospective cohort study was performed of patients presenting with bacterial or fungal keratitis at a county hospital from 2010 to 2021. Subjects were separated into 3 groups: opioid drug users (heroin, methadone, and fentanyl), nonopioid drug users, and nondrug users. Twenty-four opioid users, 77 nonopioid drug users, and 38 nondrug users were included in the study. Chi-square and t-tests were used to compare hospitalization for corneal ulcer treatment, length of hospitalization, loss to follow-up, final best-corrected visual acuity (BCVA), medication noncompliance, time to ulcer resolution, and visual disability (defined by either the legal limit for driving in California or the federal limit for blindness). Results: Opioid users had higher rates of unemployment (P = 0.002), homelessness (P = 0.018), and psychiatric conditions (P = 0.024) compared with nonopioid and nondrug users. They had more severe presentations, with worse initial BCVA of the affected eye (P = 0.003), larger ulcer size (P = 0.023), and higher rates of individuals below the legal vision thresholds for driving (P = 0.009) and blindness (P = 0.033) at initial presentation. Opioid use was associated with increased rates of hospitalization (P < 0.001), higher fortified antibiotic use (P = 0.009), worse final BCVA of the affected eye (P = 0.020), and increased rates of BCVA worse than the legal vision thresholds for driving (P = 0.043) and blindness (P < 0.001) on final presentation. Conclusions: Infectious keratitis associated with opioid use is associated with more severe presentations and poorer outcomes, including higher rates of visual disability.

Dry eye syndrome, a form of ocular surface inflammation, and chronic ocular pain are common conditions impacting activities of daily living and quality of life. Oxytocin and secretin are peptide hormones that have been shown to synergistically reduce inflammation in various tissues and attenuate the pain response at both the neuron and brain level. The oxytocin receptor (OXTR) and secretin receptor (SCTR) have been found in a wide variety of tissues and organs, including the eye. We reviewed the current literature of in vitro experiments, animal models, and human studies that examine the anti-inflammatory and anti-nociceptive roles of oxytocin and secretin. This review provides an overview of the evidence supporting oxytocin and secretin as the basis for novel treatments of dry eye and ocular pain syndromes.