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.

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.

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.

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).

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.

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.

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.

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.

AcanR3990 qPCR: A Novel, Highly Sensitive, Bioinformatically-Informed Assay to Detect Angiostrongylus cantonensis Infections.

BACKGROUND: Angiostrongylus cantonensis (Ac), or the rat lungworm, is a major cause of eosinophilic meningitis. Humans are infected by ingesting the 3rd stage larvae from primary hosts, snails, and slugs, or paratenic hosts. The currently used molecular test is a qPCR assay targeting the ITS1 rDNA region (ITS1) of Ac. METHODS: In silico design of a more sensitive qPCR assay was performed based on tandem repeats predicted to be the most abundant by the RepeatExplorer algorithm. Genomic DNA (gDNA) of Ac were used to determine the analytical sensitivity and specificity of the best primer/probe combination. This assay was then applied to clinical and environmental samples. RESULTS: The limit of detection of the best performing assay, AcanR3990, was 1 fg (the DNA equivalent of 1/100 000 dilution of a single 3rd stage larvae). Out of 127 CDC archived CSF samples from varied geographic locations, the AcanR3990 qPCR detected the presence of Ac in 49/49 ITS1 confirmed angiostrongyliasis patients, along with 15/73 samples previously negative by ITS1 qPCR despite strong clinical suspicion for angiostrongyliasis. Intermediate hosts (gastropods) and an accidental host, a symptomatic horse, were also tested with similar improvement in detection observed. AcanR3990 qPCR did not cross-react in 5 CSF from patients with proven neurocysticercosis, toxocariasis, gnathostomiasis, and baylisascariasis. AcanR3990 qPCR failed to amplify genomic DNA from the other related Angiostrongylus species tested except for Angiostrongylus mackerrasae (Am), a neurotropic species limited to Australia that would be expected to present with a clinical syndrome indistinguishable from Ac. CONCLUSION: These results suggest AcanR3990 qPCR assay is highly sensitive and specific with potential wide applicability as a One Health detection method for Ac and Am.

RPAcan3990: an Ultrasensitive Recombinase Polymerase Assay To Detect Angiostrongylus cantonensis DNA.

Angiostrongylus cantonensis is one of the leading causes of eosinophilic meningitis worldwide. A field-deployable molecular detection method could enhance both environmental surveillance and clinical diagnosis of this emerging pathogen. Accordingly, RPAcan3990, a recombinase polymerase assay (RPA), was developed to target a region predicted to be highly repeated in the A. cantonensis genome. The assay was then adapted to produce a visually interpretable fluorescent readout using an orange camera lens filter and a blue light. Using A. cantonensis genomic DNA, the limit of detection was found to be 1 fg/µl by both fluorometer measurement and visual reading. All clinical samples known to be positive for A. cantonensis from various areas of the globe were positive by RPAcan3990. Cerebrospinal fluid samples from other etiologies of eosinophilic meningitis (i.e., Toxocara sp. and Gnathostoma sp.) were negative in the RPAcan3990 assay. The optimal incubation temperature range for the reaction was between 35°C and 40°C. The assay successfully detected 1 fg/µl of A. cantonensis genomic DNA after incubation at human body temperature (in a shirt pocket). In conclusion, these data suggest RPAcan3990 is potentially a point-of-contact molecular assay capable of sensitively detecting A. cantonensis by producing visually interpretable results with minimal instrumentation.

Investigation of US Cyclospora cayetanensis outbreaks in 2019 and evaluation of an improved Cyclospora genotyping system against 2019 cyclosporiasis outbreak clusters.

Cyclosporiasis is an illness characterised by watery diarrhoea caused by the food-borne parasite Cyclospora cayetanensis. The increase in annual US cyclosporiasis cases led public health agencies to develop genotyping tools that aid outbreak investigations. A team at the Centers for Disease Control and Prevention (CDC) developed a system based on deep amplicon sequencing and machine learning, for detecting genetically-related clusters of cyclosporiasis to aid epidemiologic investigations. An evaluation of this system during 2018 supported its robustness, indicating that it possessed sufficient utility to warrant further evaluation. However, the earliest version of CDC's system had some limitations from a bioinformatics standpoint. Namely, reliance on proprietary software, the inability to detect novel haplotypes and absence of a strategy to select an appropriate number of discrete genetic clusters would limit the system's future deployment potential. We recently introduced several improvements that address these limitations and the aim of this study was to reassess the system's performance to ensure that the changes introduced had no observable negative impacts. Comparison of epidemiologically-defined cyclosporiasis clusters from 2019 to analogous genetic clusters detected using CDC's improved system reaffirmed its excellent sensitivity (90%) and specificity (99%), and confirmed its high discriminatory power. This C. cayetanensis genotyping system is robust and with ongoing improvement will form the basis of a US-wide C. cayetanensis genotyping network for clinical specimens.

Evaluation of an ensemble-based distance statistic for clustering MLST datasets using epidemiologically defined clusters of cyclosporiasis.

Outbreaks of cyclosporiasis, a food-borne illness caused by the coccidian parasite Cyclospora cayetanensis have increased in the USA in recent years, with approximately 2300 laboratory-confirmed cases reported in 2018. Genotyping tools are needed to inform epidemiological investigations, yet genotyping Cyclospora has proven challenging due to its sexual reproductive cycle which produces complex infections characterized by high genetic heterogeneity. We used targeted amplicon deep sequencing and a recently described ensemble-based distance statistic that accommodates heterogeneous (mixed) genotypes and specimens with partial genotyping data, to genotype and cluster 648 C. cayetanensis samples submitted to CDC in 2018. The performance of the ensemble was assessed by comparing ensemble-identified genetic clusters to analogous clusters identified independently based on common food exposures. Using these epidemiologic clusters as a gold standard, the ensemble facilitated genetic clustering with 93.8% sensitivity and 99.7% specificity. Hence, we anticipate that this procedure will greatly complement epidemiologic investigations of cyclosporiasis.

Cutaneous microsporidiosis in an immunosuppressed patient.

Microsporidia are a group of obligate intracellular parasites that naturally infect domestic and wild animals. Human microsporidiosis is an increasingly recognized multisystem opportunistic infection. The clinical manifestations are diverse with diarrhea being the most common presenting symptom. We present a 52-year-old woman with a history of amyopathic dermatomyositis complicated by interstitial lung disease managed with mycophenolate mofetil and hydroxychloroquine who presented with a 7-month history of recurrent subcutaneous nodules as well as intermittent diarrhea and chronic sinusitis. A punch biopsy showed superficial and deep lymphocytic and granulomatous dermatitis with focal necrosis. Tissue stains for microorganisms revealed oval 1 to 3 µm spores within the necrotic areas in multiple tissue stains. Additional studies at the Centers for Disease Control and Prevention confirmed cutaneous microsporidiosis. This case is one of very few confirmed examples of cutaneous microsporidiosis reported in the literature.

Mitochondrial Junction Region as Genotyping Marker for Cyclospora cayetanensis.

Cyclosporiasis is an infection caused by Cyclospora cayetanensis, which is acquired by consumption of contaminated fresh food or water. In the United States, cases of cyclosporiasis are often associated with foodborne outbreaks linked to imported fresh produce or travel to disease-endemic countries. Epidemiologic investigation has been the primary method for linking outbreak cases. A molecular typing marker that can identify genetically related samples would be helpful in tracking outbreaks. We evaluated the mitochondrial junction region as a potential genotyping marker. We tested stool samples from 134 laboratory-confirmed cases in the United States by using PCR and Sanger sequencing. All but 2 samples were successfully typed and divided into 14 sequence types. Typing results were identical among samples within each epidemiologically defined case cluster for 7 of 10 clusters. These findings suggest that this marker can distinguish between distinct case clusters and might be helpful during cyclosporiasis outbreak investigations.

Genotyping genetically heterogeneous Cyclospora cayetanensis infections to complement epidemiological case linkage.

Sexually reproducing pathogens such as Cyclospora cayetanensis often produce genetically heterogeneous infections where the number of unique sequence types detected at any given locus varies depending on which locus is sequenced. The genotypes assigned to these infections quickly become complex when additional loci are analysed. This genetic heterogeneity confounds the utility of traditional sequence-typing and phylogenetic approaches for aiding epidemiological trace-back, and requires new methods to address this complexity. Here, we describe an ensemble of two similarity-based classification algorithms, including a Bayesian and heuristic component that infer the relatedness of C. cayetanensis infections. The ensemble requires a set of haplotypes as input and assigns arbitrary distances to specimen pairs reflecting their most likely relationships. The approach was applied to data generated from a test cohort of 88 human fecal specimens containing C. cayetanensis, including 30 from patients whose infections were associated with epidemiologically defined outbreak clusters of cyclosporiasis. The ensemble assigned specimens to plausible clusters of genetically related infections despite their complex haplotype composition. These relationships were corroborated by a significant number of epidemiological linkages (P < 0.0001) suggesting the ensemble's utility for aiding epidemiological trace-back investigations of cyclosporiasis.

Angiostrongylus cantonensis Infection of Central Nervous System, Guiana Shield.

We report a case of eosinophilic meningitis complicated by transverse myelitis caused by Angiostrongylus cantonensis in a 10-year-old boy from Brazil who had traveled to Suriname. We confirmed diagnosis by serology and real-time PCR in the cerebrospinal fluid. The medical community should be aware of angiostrongyliasis in the Guiana Shield.

Rat Lungworm Infection Associated with Central Nervous System Disease - Eight U.S. States, January 2011-January 2017.

Angiostrongyliasis is caused by infection and migration to the brain of larvae of the parasitic nematode Angiostrongylus cantonensis, or rat lungworm. Adult A. cantonensis reside in the lungs of the definitive wild rodent host, where they produce larvae passed in feces, which are then ingested by snails and slugs (gastropods). Human infection typically occurs when gastropods containing mature larvae are inadvertently ingested by humans. Although human infection often is asymptomatic or involves transient mild symptoms, larval migration to the brain can lead to eosinophilic meningitis, focal neurologic deficits, coma, and death. The majority of cases of human angiostrongyliasis occur in Asia and the Pacific Islands, including Hawaii, but autochthonous and imported cases have been reported in the continental United States (1,2), underscoring the importance of provider recognition to ensure prompt identification and treatment. The epidemiologic and clinical features of 12 angiostrongyliasis cases in the continental United States were analyzed. These cases were identified through A. cantonensis polymerase chain reaction (PCR) testing (3) of cerebrospinal fluid (CSF) submitted to CDC from within the continental United States. Six cases were likely a result of autochthonous transmission in the southern United States. All 12 patients had CSF pleocytosis and eosinophilia, consistent with eosinophilic meningitis. Health care providers need to be aware of the possibility of angiostrongyliasis in patients with eosinophilic meningitis, especially in residents in the southern United States or persons who have traveled outside the continental United States and have a history of ingestion of gastropods or contaminated raw vegetables.

Molecular detection of Cyclospora cayetanensis in human stool specimens using UNEX-based DNA extraction and real-time PCR.

Cyclospora cayetanensis is a coccidian parasite associated with diarrheal illness. In the USA, foodborne outbreaks of cyclosporiasis have been documented almost every year since the mid-1990s. The typical approach used to identify this parasite in human stools is an examination of acid-fast-stained smears under bright-field microscopy. UV fluorescence microscopy of wet mounts is more sensitive and specific than acid-fast staining but requires a fluorescence microscope with a special filter not commonly available in diagnostic laboratories. In this study, we evaluated a new DNA extraction method based on the Universal Nucleic Acid Extraction (UNEX) buffer and compared the performances of four published real-time polymerase chain reaction (PCR) assays for the specific detection of C. cayetanensis in stool. The UNEX-based method had an improved capability to recover DNA from oocysts compared with the FastDNA stool extraction method. The best-performing real-time PCR assay was a C. cayetanensis-specific TaqMan PCR that targets the 18S ribosomal RNA gene. This new testing algorithm should be useful for detection of C. cayetanensis in human stool samples.