Application of a universal parasite diagnostic test to biological specimens collected from animals
- Lane, Meredith, Kashani, Mitra, Barratt, Joel, Qvarnstrom, Yvonne, Yabsley, Michael, Garrett, Kayla, Bradbury, Richard
- Authors: Lane, Meredith , Kashani, Mitra , Barratt, Joel , Qvarnstrom, Yvonne , Yabsley, Michael , Garrett, Kayla , Bradbury, Richard
- Date: 2023
- Type: Text , Journal article
- Relation: International Journal for Parasitology: Parasites and Wildlife Vol. 20, no. (2023), p. 20-30
- Full Text:
- Reviewed:
- Description: 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. © 2022
- Authors: Lane, Meredith , Kashani, Mitra , Barratt, Joel , Qvarnstrom, Yvonne , Yabsley, Michael , Garrett, Kayla , Bradbury, Richard
- Date: 2023
- Type: Text , Journal article
- Relation: International Journal for Parasitology: Parasites and Wildlife Vol. 20, no. (2023), p. 20-30
- Full Text:
- Reviewed:
- Description: 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. © 2022
Parasitic infection surveillance in Mississippi delta children
- Bradbury, Richard, Arguello, Irene, Lane, Meredith, Cooley, Gretchen, Handali, Sukwan
- Authors: Bradbury, Richard , Arguello, Irene , Lane, Meredith , Cooley, Gretchen , Handali, Sukwan
- Date: 2020
- Type: Text , Journal article
- Relation: American Journal of Tropical Medicine and Hygiene Vol. 103, no. 3 (2020), p. 1150-1153
- Full Text:
- Reviewed:
- Description: Some recent studies suggest ongoing transmission of parasitic diseases in the American South; however, surveys in Mississippi children are lacking. We enrolled 166 children (median age 8 years, range 4–13 years) from the Mississippi Delta region and carried out multi-parallel real-time polymerase chain reaction (PCR) for Necator americanus, Ascaris lumbricoides, and Strongyloides stercoralis on their stool samples. Dried blood spots were obtained for multiplex serology antibody detection. Of 166 children, all reported having flushable toilets, 11% had soil exposure, and 34% had a pet dog or cat. None had prior diagnosis or treatment of parasitic disease. Multi-parallel real-time PCRs were negative on the 89 stool DNA extracts available for testing. Dried blood spot testing of all 166 children determined the seroprevalence of IgG antibodies to Toxocara spp. (3.6%), Cryptosporidium (2.4%), S. stercoralis, Fasciola hepatica, and Giardia duodenalis (all 0%). In conclusion, parasitic infections and exposure were scarce in this population. Larger studies of at-risk populations are needed. © 2020 by The American Society of Tropical Medicine and Hygiene. ***Please note that there are multiple authors for this article therefore only the name of the first 5 including Federation University Australia affiliate “Richard Bradbury ” is provided in this record***
- Description: This work was funded by the Centers for Disease Control and Prevention (RSB), the Blakeslee Fund for Genetics Research at Smith College (N. P. and S. A. W.), and the University of Mississippi Medical Center (Office of the Vice Chancellor for Research, the University of Mississippi Medical Center). R. B. reports a patent WO2019060840 “Removing Interfering Host NucleicAcids for Molecular Parasite Detection” with royalties paid to Centers for Disease Control and Prevention. This trial was observational and is exempt from registration at clinicaltrials.gov Disclaimer: The findings and conclusions of this work are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry. This work was presented at the American Society of Tropical Medicine and Hygiene Conference: Poster 522, October 28–November 1, New Orleans, LA
- Authors: Bradbury, Richard , Arguello, Irene , Lane, Meredith , Cooley, Gretchen , Handali, Sukwan
- Date: 2020
- Type: Text , Journal article
- Relation: American Journal of Tropical Medicine and Hygiene Vol. 103, no. 3 (2020), p. 1150-1153
- Full Text:
- Reviewed:
- Description: Some recent studies suggest ongoing transmission of parasitic diseases in the American South; however, surveys in Mississippi children are lacking. We enrolled 166 children (median age 8 years, range 4–13 years) from the Mississippi Delta region and carried out multi-parallel real-time polymerase chain reaction (PCR) for Necator americanus, Ascaris lumbricoides, and Strongyloides stercoralis on their stool samples. Dried blood spots were obtained for multiplex serology antibody detection. Of 166 children, all reported having flushable toilets, 11% had soil exposure, and 34% had a pet dog or cat. None had prior diagnosis or treatment of parasitic disease. Multi-parallel real-time PCRs were negative on the 89 stool DNA extracts available for testing. Dried blood spot testing of all 166 children determined the seroprevalence of IgG antibodies to Toxocara spp. (3.6%), Cryptosporidium (2.4%), S. stercoralis, Fasciola hepatica, and Giardia duodenalis (all 0%). In conclusion, parasitic infections and exposure were scarce in this population. Larger studies of at-risk populations are needed. © 2020 by The American Society of Tropical Medicine and Hygiene. ***Please note that there are multiple authors for this article therefore only the name of the first 5 including Federation University Australia affiliate “Richard Bradbury ” is provided in this record***
- Description: This work was funded by the Centers for Disease Control and Prevention (RSB), the Blakeslee Fund for Genetics Research at Smith College (N. P. and S. A. W.), and the University of Mississippi Medical Center (Office of the Vice Chancellor for Research, the University of Mississippi Medical Center). R. B. reports a patent WO2019060840 “Removing Interfering Host NucleicAcids for Molecular Parasite Detection” with royalties paid to Centers for Disease Control and Prevention. This trial was observational and is exempt from registration at clinicaltrials.gov Disclaimer: The findings and conclusions of this work are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry. This work was presented at the American Society of Tropical Medicine and Hygiene Conference: Poster 522, October 28–November 1, New Orleans, LA
Sensitive universal detection of blood parasites by selective pathogen-DNA enrichment and deep amplicon sequencing
- Flaherty, Briana, Barratt, Joel, Lane, Meredith, Talundzic, Eldin, Bradbury, Richard
- Authors: Flaherty, Briana , Barratt, Joel , Lane, Meredith , Talundzic, Eldin , Bradbury, Richard
- Date: 2021
- Type: Text , Journal article
- Relation: Microbiome Vol. 9, no. 1 (2021), p.
- Full Text:
- Reviewed:
- Description: 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. [MediaObject not available: see fulltext.]. © 2021, The Author(s).
- Authors: Flaherty, Briana , Barratt, Joel , Lane, Meredith , Talundzic, Eldin , Bradbury, Richard
- Date: 2021
- Type: Text , Journal article
- Relation: Microbiome Vol. 9, no. 1 (2021), p.
- Full Text:
- Reviewed:
- Description: 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. [MediaObject not available: see fulltext.]. © 2021, The Author(s).
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