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

Abstract

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.ResultsA 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 μl in infected monkey blood and 61 parasites per μl 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.ConclusionsTo 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-017-2273-7) contains supplementary material, which is available to authorized users.

Rapid method for detecting and differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in sputum by fluorescence in situ hybridization with DNA probes

“…Similarly rapid FISH assays based on the same approach, and which are suitable for use in disease-endemic countries, have also been applied for the detection and differentiation of different species of human malaria parasites in thin blood films (Shah et al, 2017b;Shah et al, 2015).…”

A Fluorescence In Situ Hybridization (FISH) Test for Diagnosing Babesiosis

“…The Babesia genus-specific test was performed according to the manufacturer’s instructions provided with the kit similar to the FISH test procedures for Plasmodium falciparum and Plasmodium vivax [ 9 ] and Plasmodium knowlesi [ 10 ]. Three parts EDTA-treated whole blood or fresh cultures for testing were first mixed with 1 part SPR by volume.…”

“…The analytical sensitivity was measured as the limit of detection (LOD) as previously described for FISH tests on P. falciparum , P. vivax , and P. knowlesi [ 9 , 10 ]. The LOD was determined as the lowest concentration of parasites that could be detected by FISH in every one of 20 replicate smears prepared as described above at different serial dilutions of B. duncani (ATCC PRA-302) and B. microti (ATCC 30221D) grown in hamster blood provided by Dr. Alan Ashbaugh, University of Cincinnati, OH, USA.…”

“…Common laboratory tests for diagnosing babesiosis depend on detecting (i) parasites in stained blood smears by microscopy, (ii) serum antibodies by immunoassays, and (iii) Babesia nucleic acid in blood after PCR amplification [ 3 , 4 ]. Fluorescence in situ hybridization (FISH) tests are used for detecting malaria parasites in blood smears [ 9 , 10 ] and tuberculosis bacilli in sputum smears [ 11 , 12 ]. We now report the details of a commercially available FISH test designed to detect all known species of Babesia parasites in blood smears.…”

Application of Molecular Pathology Techniques to Understand Mechanisms of Disease in Smallmouth Bass

“…In situ hybridization allows for the detection and visualization of DNA and RNA within tissue sections (Cassidy and Jones 2014). With FISH, probes can be labeled with different fluorescence tags to detect multiple targets in one tissue sample (Shah et al 2017). Performing FISH with probes designed to hybridize to bacteria, such as Flavobacterium spp.…”