PMID- 28724402 OWN - NLM STAT- MEDLINE DCOM- 20180319 LR - 20220129 IS - 1756-3305 (Electronic) IS - 1756-3305 (Linking) VI - 10 IP - 1 DP - 2017 Jul 19 TI - A dual colour fluorescence in situ hybridization (FISH) assay for identifying the zoonotic malaria parasite Plasmodium knowlesi with a potential application for the specific diagnosis of knowlesi malaria in peripheral-level laboratories of Southeast Asia. PG - 342 LID - 10.1186/s13071-017-2273-7 [doi] LID - 342 AB - BACKGROUND: Plasmodium knowlesi is primarily responsible for zoonotic malaria in several Southeast Asian countries. Precise identification of the parasite in the blood of patients presently relies on an expensive and elaborate PCR procedure because microscopic examination of blood and other available field identification techniques lack adequate specificity. Therefore, the use of a simple and inexpensive dual-colour fluorescence in situ hybridization (FISH) assay, analogous to FISH assays recently described for Plasmodium falciparum and Plasmodium vivax, was investigated as a potential tool for identifying P. knowlesi. RESULTS: A P. knowlesi 18S rDNA sequence-based DNA probe was used to test thin blood smears of P. knowlesi by FISH, and fluorescence viewed in a light microscope fitted with a light emitting diode light source and appropriate emission and barrier filters. The limit of detection in the P. knowlesi FISH assay was 84 parasites per mul in infected monkey blood and 61 parasites per mul for P. knowlesi cultured in human blood. The P. knowlesi-specific FISH probe detected only P. knowlesi and not P. falciparum, Plasmodium malariae, Plasmodium ovale, P. vivax or a panel of other human blood-borne pathogens. A previously described Plasmodium genus-specific probe used simultaneously in the P. knowlesi FISH assay reacted with all tested Plasmodium species. CONCLUSIONS: To our knowledge, this is the first description of a FISH assay for P. knowlesi that is potentially useful for diagnosing infections in remote laboratories in endemic countries. FAU - Shah, Jyotsna AU - Shah J AD - ID-FISH Technology, Palo Alto, CA, USA. jyotsna@aol.com. AD - IGeneX, Palo Alto, CA, USA. jyotsna@aol.com. FAU - Poruri, Akhila AU - Poruri A AD - ID-FISH Technology, Palo Alto, CA, USA. AD - IGeneX, Palo Alto, CA, USA. FAU - Mark, Olivia AU - Mark O AD - ID-FISH Technology, Palo Alto, CA, USA. AD - IGeneX, Palo Alto, CA, USA. FAU - Khadilka, Urmila AU - Khadilka U AD - Kasturba Medical College Hospital, Mangalore, India. FAU - Mohring, Franziska AU - Mohring F AD - London School of Hygiene and Tropical Medicine, Keppel Street, London, UK. FAU - Moon, Robert W AU - Moon RW AD - London School of Hygiene and Tropical Medicine, Keppel Street, London, UK. FAU - Ramasamy, Ranjan AU - Ramasamy R AUID- ORCID: 0000-0003-0246-7053 AD - ID-FISH Technology, Palo Alto, CA, USA. rjr200911@yahoo.com. LA - eng GR - MR/M021157/1/MRC_/Medical Research Council/United Kingdom GR - R21 CA094303/CA/NCI NIH HHS/United States GR - R33 CA094303/CA/NCI NIH HHS/United States PT - Journal Article DEP - 20170719 PL - England TA - Parasit Vectors JT - Parasites & vectors JID - 101462774 RN - 0 (DNA, Protozoan) RN - 0 (DNA, Ribosomal) RN - 0 (Oligonucleotide Probes) RN - 0 (RNA, Ribosomal, 18S) SB - IM MH - Asia, Southeastern MH - DNA, Protozoan/chemistry/genetics MH - DNA, Ribosomal/chemistry/genetics MH - Humans MH - In Situ Hybridization, Fluorescence/*methods MH - Malaria/*diagnosis/parasitology MH - Molecular Diagnostic Techniques/*methods MH - Oligonucleotide Probes/genetics MH - Parasitemia/*diagnosis/parasitology MH - Plasmodium knowlesi/genetics/*isolation & purification MH - RNA, Ribosomal, 18S/genetics MH - Sequence Analysis, DNA PMC - PMC5517825 OTO - NOTNLM OT - DNA probe OT - Fluorescence in situ hybridization OT - Malaria diagnosis OT - Plasmodium knowlesi OT - Zoonotic malaria COIS- ETHICS APPROVAL: The study was reviewed and approved by the Institutional Review Boards of Kasturba Medical College, Mangalore, India and the Kenya Medical Research Institute/Walter Reed project, Kisumu, Kenya. Approval was granted to ID-FISH Technology Inc., for the use of archived de-identified samples submitted for routine testing that would otherwise have been discarded. CONSENT FOR PUBLICATION: Not applicable. COMPETING INTERESTS: JS is the Founder and Chief Scientific Officer of ID-FISH and owns ID-FISH stock. OM is an employee and RR an affiliate of ID-FISH. Other authors have no affiliation with ID-FISH. JS and OM are inventors on the following relevant patents: Nucleic acid probes and methods for detecting Plasmodium parasites. Patent number US 007927801, issue date 4/19/2011; Nucleic acid probes and methods for detecting Plasmodium parasites (54) (75) Patent number US 008323901, Issue Date 12/4/2012; Nucleic acid probes and methods for detecting Plasmodium parasites (54) (75) Patent number US 008592568B2, Issue Date 11/26/2013. The PK-FISH assay kit is proprietary to ID-FISH and subject to US patent application number 15/389,827 on nucleic acid probes and methods of detection of Plasmodium knowlesi. PUBLISHER'S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EDAT- 2017/07/21 06:00 MHDA- 2018/03/20 06:00 PMCR- 2017/07/19 CRDT- 2017/07/21 06:00 PHST- 2017/01/10 00:00 [received] PHST- 2017/07/05 00:00 [accepted] PHST- 2017/07/21 06:00 [entrez] PHST- 2017/07/21 06:00 [pubmed] PHST- 2018/03/20 06:00 [medline] PHST- 2017/07/19 00:00 [pmc-release] AID - 10.1186/s13071-017-2273-7 [pii] AID - 2273 [pii] AID - 10.1186/s13071-017-2273-7 [doi] PST - epublish SO - Parasit Vectors. 2017 Jul 19;10(1):342. doi: 10.1186/s13071-017-2273-7.