Evaluation of coccidia DNA in irrigation pond water and wastewater sludge associated with Cyclospora cayetanensis 18S rRNA gene qPCR detections.

The coccidian parasite Cyclospora cayetanensis is the causative agent for foodborne outbreaks of cyclosporiasis disease and multiple annual fresh produce recalls. The aim of this study was to identify potential cross-reacting species for the C. cayetanensis 18S rRNA and MIT1C gene target real-time quantitative polymerase chain reaction (qPCR) assays. The environmental samples evaluated were irrigation pond water, produce wash water, and wastewater treatment sludge from a previous study with qPCR detections of C. cayetanensis by the 18S rRNA gene target qPCR. From these samples, longer regions of the 18S rRNA gene and the mitochondrial cytochrome c oxidase subunit III gene (cox3) were sequenced. Of 65 irrigation pond water samples with positive test results using the C. cayetanensis 18S rRNA gene qPCR assay, none had MIT1C qPCR assay detections or sequences that clustered with C. cayetanensis based on sequencing of the cox3 and 18S rRNA gene. Sequences from these samples clustered around coccidia sequences found in bird, fish, reptile, and amphibian hosts. Of 26 sludge samples showing detections by either qPCR assay, 14 (54%) could be confirmed as containing C. cayetanensis by sequencing of cox3 and 18S rRNA gene regions. In three of the remaining sludge samples, sequenced reads clustered with coccidia from rodents. This study demonstrated that caution should be taken when interpreting qPCR C. cayetanensis detection data in environmental samples and sequencing steps will likely be needed for confirmation. IMPORTANCE: Fresh produce is a leading transmission source in cyclosporiasis outbreaks. It is therefore essential to understand the role that produce-growing environments play in the spread of this disease. To accomplish this, sensitive and specific tests for environmental and irrigation waters must be developed. Potential cross-reactions of Cyclospora cayetanensis real-time quantitative polymerase chain reaction (qPCR) assays have been identified, hindering the ability to accurately identify this parasite in the environment. Amplicon sequencing of the cox3 and 18S rRNA genes revealed that all irrigation pond water and two sludge samples that initially detected C. cayetanensis by qPCR were most likely cross-reactions with related coccidian organisms shed from birds, fish, reptiles, amphibians, and rodents. These results support that a single testing method for environmental samples is likely not adequate for sensitive and specific detection of C. cayetanensis.

Outbreak of Human Trichinellosis - Arizona, Minnesota, and South Dakota, 2022.

Trichinellosis is a parasitic zoonotic disease transmitted through the consumption of meat from animals infected with Trichinella spp. nematodes. In North America, human trichinellosis is rare and is most commonly acquired through consumption of wild game meat. In July 2022, a hospitalized patient with suspected trichinellosis was reported to the Minnesota Department of Health. One week before symptom onset, the patient and eight other persons shared a meal that included bear meat that had been frozen for 45 days before being grilled and served rare with vegetables that had been cooked with the meat. Investigation identified six trichinellosis cases, including two in persons who consumed only the vegetables. Motile Trichinella larvae were found in remaining bear meat that had been frozen for >15 weeks. Molecular testing identified larvae from the bear meat as Trichinella nativa, a freeze-resistant species. Persons who consume meat from wild game animals should be aware that that adequate cooking is the only reliable way to kill Trichinella parasites and that infected meat can cross-contaminate other foods.

Outbreak of Human Trichinellosis - Arizona, Minnesota, and South Dakota, 2022.

Trichinellosis is a parasitic zoonotic disease transmitted through the consumption of meat from animals infected with Trichinella spp. nematodes. In North America, human trichinellosis is rare and is most commonly acquired through consumption of wild game meat. In July 2022, a hospitalized patient with suspected trichinellosis was reported to the Minnesota Department of Health. One week before symptom onset, the patient and eight other persons shared a meal that included bear meat that had been frozen for 45 days before being grilled and served rare with vegetables that had been cooked with the meat. Investigation identified six trichinellosis cases, including two in persons who consumed only the vegetables. Motile Trichinella larvae were found in remaining bear meat that had been frozen for >15 weeks. Molecular testing identified larvae from the bear meat as Trichinella nativa, a freeze-resistant species. Persons who consume meat from wild game animals should be aware that that adequate cooking is the only reliable way to kill Trichinella parasites and that infected meat can cross-contaminate other foods.

Genetic characterization and description of Leishmania (Leishmania) ellisi sp. nov.: a new human-infecting species from the USA.

In a 2018 report, an unusual case of cutaneous leishmaniasis was described in a 72-year-old female patient residing in Arizona, United States of America (USA). Preliminary analysis of the 18S rDNA and glyceraldehyde-3-phosphate dehydrogenase genes supported the conclusion that the Leishmania strain (strain 218-L139) isolated from this case was a novel species, though a complete taxonomic description was not provided. Identification of Leishmania at the species level is critical for clinical management and epidemiologic investigations so it is important that novel human-infecting species are characterized taxonomically and assigned a unique scientific name compliant with the ICZN code. Therefore, we sought to provide a complete taxonomic description of Leishmania strain 218-L139. Phylogenetic analysis of several nuclear loci and partial maxicircle genome sequences supported its position within the subgenus Leishmania and further clarified the distinctness of this new species. Morphological characterization of cultured promastigotes and amastigotes from the original case material is also provided. Thus, we conclude that Leishmania (Leishmania) ellisi is a new cause of autochthonous cutaneous leishmaniasis in the USA.

Simultaneous targeted amplicon deep sequencing and library preparation for a time and cost-effective universal parasite diagnostic sequencing approach.

We recently described a targeted amplicon deep sequencing (TADS) strategy that utilizes a nested PCR targeting the 18S rDNA gene of blood-borne parasites. The assay facilitates selective digestion of host DNA by targeting enzyme restriction sites present in vertebrates but absent in parasites. This enriching of parasite-derived amplicon drastically reduces the proportion of host-derived reads during sequencing and results in the sensitive detection of several clinically important blood parasites including Plasmodium spp., Babesia spp., kinetoplastids, and filarial nematodes. Despite these promising results, high costs and the laborious nature of metagenomics sequencing are prohibitive to the routine use of this assay in most laboratories. We describe and evaluate a new metagenomic approach that utilizes a set of primers modified from our original assay that incorporates Illumina barcodes and adapters during the PCR steps. This modification makes amplicons immediately compatible with sequencing on the Illumina MiSeq platform, removing the need for a separate library preparation, which is expensive and time-consuming. We compared this modified assay to our previous nested TADS assay in terms of preparation speed, limit of detection (LOD), and cost. Our modifications reduced assay turnaround times from 7 to 5 days. The cost decreased from approximately $40 per sample to $11 per sample. The modified assay displayed comparable performance in the detection and differentiation of human-infecting Plasmodium spp., Babesia spp., kinetoplastids, and filarial nematodes in clinical samples. The LOD of this modified approach was determined for malaria parasites and remained similar to that previously reported for our earlier assay (0.58 Plasmodium falciparum parasites/µL of blood). These modifications markedly reduced costs and turnaround times, making the assay more amenable to routine diagnostic applications.

Cross-Sectional Study of Soil-Transmitted Helminthiases in Black Belt Region of Alabama, USA.

We conducted a cross-sectional study to determine the prevalence of soil-transmitted helminthiases (STH) in areas of rural Alabama, USA, that have sanitation deficits. We enrolled 777 children; 704 submitted stool specimens and 227 a dried blood spot sample. We microscopically examined stool specimens from all 704 children by using Mini-FLOTAC for helminth eggs. We tested a subset by using molecular techniques: real-time PCR analysis for 5 STH species, TaqMan Array Cards for enteric helminths, and digital PCR for Necator americanus hookworm. We analyzed dried blood spots for Strongyloides stercoralis and Toxocara spp. roundworms by using serologic testing. Despite 12% of our cohort reporting living in homes that directly discharge untreated domestic wastewater, stool testing for STH was negative; however, 5% of dried blood spots were positive for Toxocara spp. roundworms. Survey data suggests substantial numbers of children in this region may be exposed to raw sewage, which is itself a major public health concern.

Surveillance for Soil-Transmitted Helminths in High-Risk County, Mississippi, USA.

Recent reports of hookworm infection in Alabama, USA, has prompted surveillance in Mississippi, given the states' similar environmental conditions. We collected stool specimens from 277 children in Rankin County, Mississippi. Kato-Katz microscopic smear, agar plate culture, and quantitative PCR indicated no soil-transmitted helminths. Nevertheless, further surveillance in other high-risk Mississippi counties is warranted.

Comparison of two novel one-tube nested real-time qPCR assays to detect human-infecting Cyclospora spp.

IMPORTANCE: Human-infecting Cyclospora spp. cause gastrointestinal distress among healthy individuals contributing to morbidity and putting stress on the economics of countries and companies in the form of produce recalls. Accessible and easy-to-use diagnostic tools available to a wide variety of laboratories would aid in the early detection of possible outbreaks of cyclosporiasis. This, in turn, will assist in the timely traceback investigation to the suspected source of an outbreak by informing the smallest possible recall and protecting consumers from contaminated produce. This manuscript describes two novel detection methods with improved performance for the causative agents of cyclosporiasis when compared to the currently used 18S assay.

Novel insights on the genetic population structure of human-infecting Cyclospora spp. and evidence for rapid subtype selection among isolates from the USA.

Human-infecting Cyclospora was recently characterized as three species, two of which (C. cayetanensis and C. ashfordi) are currently responsible for all known human infections in the USA, yet much remains unknown about the genetic structure within these two species. Here, we investigate Cyclospora genotyping data from 2018 through 2022 to ascertain if there are temporal patterns in the genetic structure of Cyclospora parasites that cause infections in US residents from year to year. First, we investigate three levels of genetic characterization: species, subpopulation, and strain, to elucidate annual trends in Cyclospora infections. Next, we determine if shifts in genetic diversity can be linked to any of the eight loci used in our Cyclospora genotyping approach. We observed fluctuations in the abundance of Cyclospora types at the species and subpopulation levels, but no significant temporal trends were identified; however, we found recurrent and sporadic strains within both C. ashfordi and C. cayetanensis. We also uncovered major shifts in the mitochondrial genotypes in both species, where there was a universal increase in abundance of a specific mitochondrial genotype that was relatively abundant in 2018 but reached near fixation (was observed in over 96% of isolates) in C. ashfordi by 2022. Similarly, this allele jumped from 29% to 82% relative abundance of isolates belonging to C. cayetanensis. Overall, our analysis uncovers previously unknown temporal-genetic patterns in US Cyclospora types from 2018 through 2022 and is an important step to presenting a clearer picture of the factors influencing cyclosporiasis outbreaks in the USA.

The limit of detection of the BioFire® FilmArray® gastrointestinal panel for the foodborne parasite Cyclospora cayetanensis.

Cyclosporiasis is a foodborne diarrheal illness caused by the parasite Cyclospora cayetanensis. The BioFire® FilmArray® gastrointestinal (FilmArray GI) panel is a common method for diagnosing cyclosporiasis from clinical stool samples. The currently published limit of detection (LOD) of this panel is in genome equivalents; however, it is unclear how this relates to the number of C. cayetanensis oocysts in a clinical sample. In this study, we developed a technique to determine the LOD in terms of oocysts, using a cell sorter to sort 1 to 50 C. cayetanensis oocyst(s) previously purified from three human stool sources. We found the FilmArray GI panel detected samples with ≥20 C. cayetanensis oocysts in 100% of replicates, with varying detection among samples with 1, 5, or 10 C. cayetanensis oocysts. This method provides a parasitologically relevant LOD that should enable comparison among C. cayetanensis detection techniques, including the FilmArray GI panel.

Retrospective evaluation of an integrated molecular-epidemiological approach to cyclosporiasis outbreak investigations - United States, 2021.

Cyclosporiasis results from an infection of the small intestine by Cyclospora parasites after ingestion of contaminated food or water, often leading to gastrointestinal distress. Recent developments in temporally linking genetically related Cyclospora isolates demonstrated effectiveness in supporting epidemiological investigations. We used 'temporal-genetic clusters' (TGCs) to investigate reported cyclosporiasis cases in the United States during the 2021 peak-period (1 May - 31 August 2021). Our approach split 655 genotyped isolates into 55 genetic clusters and 31 TGCs. We linked two large multi-state epidemiological clusters (Epidemiologic Cluster 1 [n = 136 cases, 54 genotyped] and Epidemiologic Cluster 2 [n = 42 cases, 15 genotyped]) to consumption of lettuce varieties; however, product traceback did not identify a specific product for either cluster due to the lack of detailed product information. To evaluate the utility of TGCs, we performed a retrospective case study comparing investigation outcomes of outbreaks first detected using epidemiological methods with those of the same outbreaks had TGCs been used to first detect them. Our study results indicate that adjustments to routine epidemiological approaches could link additional cases to epidemiological clusters of cyclosporiasis. Overall, we show that CDC's integrated genotyping and epidemiological investigations provide valuable insights into cyclosporiasis outbreaks in the United States.

Genetic characterization of Strongyloides fuelleborni infecting free-roaming African vervets (Chlorocebus aethiops sabaeus) on the Caribbean island of St. Kitts.

Human strongyloidiasis is an important neglected tropical disease primarily caused by the nematode Strongyloides stercoralis, and to a lesser extent Strongyloides fuelleborni which mainly infects non-human primates. Zoonotic sources of infection have important implications for control and prevention of morbidity and mortality caused by strongyloidiasis. Recent molecular evidence suggests that for S. fuelleborni, primate host specificity is variable among genotypes across the Old World, and consequently that these types likely vary in their capacity for human spillover infections. Populations of free-roaming vervet monkeys (Chlorocebus aethiops sabaeus), introduced to the Caribbean Island of Staint Kitts from Africa, live in close contact with humans, and concern has arisen regarding their potential to serve as reservoirs of zoonotic infections. In this study, we sought to determine the genotypes of S. fuelleborni infecting St. Kitts vervets to explore whether they are potential reservoirs for human-infecting S. fuelleborni types. Fecal specimens were collected from St. Kitts vervets and S. fuelleborni infections were confirmed microscopically and by PCR. Strongyloides fuelleborni genotypes were determined from positive fecal specimens using an Illumina amplicon sequencing-based genotyping approach targeting the mitochondrial cox1 locus and 18S rDNA hypervariable regions I and IV of Strongyloides species. Phylogenetic analysis of resultant genotypes supported that S. fuelleborni from St. Kitts vervets is of an exclusively African variety, falling within the same monophyletic group as an isolate which has been detected previously in a naturally infected human from Guinea-Bissau. This observation highlights that St. Kitts vervets may serve as potential reservoirs for zoonotic S. fuelleborni infection, which warrants further exploration.

Application of a universal parasite diagnostic test to biological specimens collected from animals.

A previously described universal parasite diagnostic (nUPDx) based on PCR amplification of the 18S rDNA and deep-amplicon sequencing, can detect human blood parasites with a sensitivity comparable to real-time PCR. To date, the efficacy of this assay has only been assessed on human blood. This study assessed the utility of nUPDx for the detection of parasitic infections in animals using blood, tissues, and other biological sample types from mammals, birds, and reptiles, known to be infected with helminth, apicomplexan, or pentastomid parasites (confirmed by microscopy or PCR), as well as negative samples. nUPDx confirmed apicomplexan and/or nematode infections in 24 of 32 parasite-positive mammals, while also identifying several undetected coinfections. nUPDx detected infections in 6 of 13 positive bird and 1 of 2 positive reptile samples. When applied to 10 whole parasite specimens (worms and arthropods), nUPDx identified all to the genus or family level, and detected one incorrect identification made by morphology. Babesia sp. infections were detected in 5 of the 13 samples that were negative by other diagnostic approaches. While nUPDx did not detect PCR/microscopy-confirmed trichomonads or amoebae in cloacal swabs/tissue from 8 birds and 2 reptiles due to primer template mismatches, 4 previously undetected apicomplexans were detected in these samples. Future efforts to improve the utility of the assay should focus on validation against a larger panel of tissue types and animal species. Overall, nUPDx shows promise for use in both veterinary diagnostics and wildlife surveillance, especially because species-specific PCRs can miss unknown or unexpected pathogens.

The Epidemiology and Clinical Features of Non-Keratitis Acanthamoeba Infections in the United States, 1956-2020.

Background: Acanthamoeba is a free-living ameba that can cause severe disease affecting the central nervous system, skin, sinuses, and other organs, particularly in immunocompromised individuals. These rare but severe infections are often fatal, yet incompletely described. Methods: Cases included were either reported to the Centers for Disease Control and Prevention (CDC) Free-Living Ameba program or published in scientific literature. Characteristics of all patients in the United States with laboratory-confirmed non-keratitis Acanthamoeba infections were described using descriptive statistics, and associations with survival were determined using χ2 and Fisher exact tests. Results: Of 173 patients identified, 71% were male and the median age was 44 years (range, 0-87 years). Of these, 26 (15%) survived. Most patients (88%) had at least 1 immunocompromising condition, most commonly human immunodeficiency virus (39%), cancer (28%), and solid organ or hematopoietic stem cell transplant (28%). Granulomatous amebic encephalitis (GAE) was the most common disease presentation (71%). Skin (46%), sinuses (29%), lungs (13%), and bone (6%) were also involved. Nearly half of patients (47%) had involvement of >1 organ system. Survival was less frequent among those with GAE (3%, P < .001) compared with cutaneous disease, rhinosinusitis, or multiorgan disease not including GAE. Of 7 who received the currently recommended treatment regimen, 5 (71%) survived. Conclusions: Non-keratitis Acanthamoeba infections occur primarily in immunocompromised individuals and are usually fatal. Survival may be associated with disease presentation and treatment. Providers who care for at-risk patients should be aware of the various disease manifestations to improve early recognition and treatment.

A Pilot Comparison of Fixatives for Hookworm Real-time Polymerase Chain Reaction.

Polymerase chain reaction (PCR) is increasingly used in the diagnosis of soil-transmitted helminth infections. Despite this, few studies have evaluated the impact of different fecal fixatives on the outcome of fecal helminth qPCR analysis, and none have evaluated the effect of commercial parasitology fixatives commonly used in diagnostic laboratories. We fixed dog feces containing Ancylostoma spp. hookworm eggs in zinc polyvinyl alcohol (Zn-PVA) and Total-Fix, and with 70% ethanol (EtOH) as a control. DNA was extracted at timepoints 11, 33, 64, and 94 days and subjected to Ancylostoma spp. quantitative PCR (qPCR). A linear regression model was created to assess the effect of preservative types on the temporal change of qPCR quantification cycle number (Cq) values, accounting for variances among individual animals. Fixation in 70% EtOH least affected Cq values over 94 days. Total-Fix preservation yielded a higher Cq overall, but there was no significant difference compared with 70% EtOH fixation. Fixation in Zn-PVA resulted in significantly (P < 0.001) higher Cq values than 70% EtOH after only 33 days and loss of amplification at 64 days. Consistent with other helminth fixation studies, 70% EtOH performed well in preserving hookworm DNA over 94 days. Total-Fix provided a comparable alternative for qPCR analysis for hookworm. Fixation in Zn-PVA resulted in loss of detectable hookworm DNA at 64 days, as determined by qPCR.

Cyclospora cayetanensis comprises at least 3 species that cause human cyclosporiasis.

The apicomplexan parasite Cyclospora cayetanensis causes seasonal foodborne outbreaks of the gastrointestinal illness cyclosporiasis. Prior to the coronavirus disease-2019 pandemic, annually reported cases were increasing in the USA, leading the US Centers for Disease Control and Prevention to develop a genotyping tool to complement cyclosporiasis outbreak investigations. Thousands of US isolates and 1 from China (strain CHN_HEN01) were genotyped by Illumina amplicon sequencing, revealing 2 lineages (A and B). The allelic composition of isolates was examined at each locus. Two nuclear loci (CDS3 and 360i2) distinguished lineages A and B. CDS3 had 2 major alleles: 1 almost exclusive to lineage A and the other to lineage B. Six 360i2 alleles were observed ­ 2 exclusive to lineage A (alleles A1 and A2), 2 to lineage B (B1 and B2) and 1 (B4) was exclusive to CHN_HEN01 which shared allele B3 with lineage B. Examination of heterozygous genotypes revealed that mixtures of A- and B-type 360i2 alleles occurred rarely, suggesting a lack of gene flow between lineages. Phylogenetic analysis of loci from whole-genome shotgun sequences, mitochondrial and apicoplast genomes, revealed that CHN_HEN01 represents a distinct lineage (C). Retrospective examination of epidemiologic data revealed associations between lineage and the geographical distribution of US infections plus strong temporal associations. Given the multiple lines of evidence for speciation within human-infecting Cyclospora, we provide an updated taxonomic description of C. cayetanensis, and describe 2 novel species as aetiological agents of human cyclosporiasis: Cyclospora ashfordi sp. nov. and Cyclospora henanensis sp. nov. (Apicomplexa: Eimeriidae).

Neuroinvasive Onchocerca lupi Infection in a Ten-Year-Old Girl.

The nematode Onchocerca lupi is an emerging human pathogen. Though its life cycle is not well studied, it likely infects humans after a bite from a black fly vector, which in turn acquires infective microfilariae from an infected canid. These microfilariae mature into an infective larval stage within the fly. Among six reported cases in the United States, five involved children, and all occurred in the southwest. In this report, we present a case of O. lupi infection with cervical spine invasion in a healthy 10-year-old girl. She presented with five months of neurological symptoms from a rural and medically underserved area, highlighting a need for clinical vigilance in such settings for this emerging infectious threat in the American southwest.

Evaluation of various distance computation methods for construction of haplotype-based phylogenies from large MLST datasets.

Multi-locus sequence typing (MLST) is widely used to investigate genetic relationships among eukaryotic taxa, including parasitic pathogens. MLST analysis workflows typically involve construction of alignment-based phylogenetic trees - i.e., where tree structures are computed from nucleotide differences observed in a multiple sequence alignment (MSA). Notably, alignment-based phylogenetic methods require that all isolates/taxa are represented by a single sequence. When multiple loci are sequenced these sequences may be concatenated to produce one tree that includes information from all loci. Alignment-based phylogenetic techniques are robust and widely used yet possess some shortcomings, including how heterozygous sites are handled, intolerance for missing data (i.e., partial genotypes), and differences in the way insertions-deletions (indels) are scored/treated during tree construction. In certain contexts, 'haplotype-based' methods may represent a viable alternative to alignment-based techniques, as they do not possess the aforementioned limitations. This is namely because haplotype-based methods assess genetic similarity based on numbers of shared (i.e., intersecting) haplotypes as opposed to similarities in nucleotide composition observed in an MSA. For haplotype-based comparisons, choosing an appropriate distance statistic is fundamental, and several statistics are available to choose from. However, a comprehensive assessment of various available statistics for their ability to produce a robust haplotype-based phylogenetic reconstruction has not yet been performed. We evaluated seven distance statistics by applying them to extant MLST datasets from the gastrointestinal parasite Cyclospora cayetanensis and two species of pathogenic nematode of the genus Strongyloides. We compare the genetic relationships identified using each statistic to epidemiologic, geographic, and host metadata. We show that Barratt's heuristic definition of genetic distance was the most robust among the statistics evaluated. Consequently, it is proposed that Barratt's heuristic represents a useful approach for use in the context of challenging MLST datasets possessing features (i.e., high heterozygosity, partial genotypes, and indel or repeat-based polymorphisms) that confound or preclude the use of alignment-based methods.

Genotyping Cyclospora cayetanensis From Multiple Outbreak Clusters With An Emphasis on a Cluster Linked to Bagged Salad Mix-United States, 2020.

Cyclosporiasis is a diarrheal illness caused by the foodborne parasite Cyclospora cayetanensis. Annually reported cases have been increasing in the United States prompting development of genotyping tools to aid cluster detection. A recently developed Cyclospora genotyping system based on 8 genetic markers was applied to clinical samples collected during the cyclosporiasis peak period of 2020, facilitating assessment of its epidemiologic utility. While the system performed well and helped inform epidemiologic investigations, inclusion of additional markers to improve cluster detection was supported. Consequently, investigations have commenced to identify additional markers to enhance performance.