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

Abstract

Apicomplexan parasites of the genus Babesia cause babesiosis in humans and animals. The microscopic examination of stained blood smears, detection of serum antibodies by immunoassays, and PCR-based identification of parasite nucleic acid in blood are common laboratory methods for diagnosing babesiosis. The present study evaluated a commercially available Babesia genus-specific fluorescence in situ hybridization (FISH) test for detecting Babesia parasites in blood smears. The FISH test detected Babesia duncani and Babesia microti, two common species that cause human infections in the USA, and other Babesia species of human and veterinary importance in less than two hours. The Babesia genus-specific FISH test supplements other existing laboratory methods for diagnosing babesiosis and may be particularly useful in resource-limited laboratories.

Combined Immunofluorescence (IFA) and Fluorescence In Situ Hybridization (FISH) Assays for Diagnosing Babesiosis in Patients from the USA, Europe and Australia

“…The Babesia genus-specific FISH assay was performed on blood smears on glass microscope slides using a kit (catalogue number BabGK04 from ID-FISH Technology Inc., Milpitas, CA, USA) according to the manufacturer’s instructions as described previously in detail [ 9 ]. Fluorescence was then viewed using a light microscope with an LED attachment containing 492 nm excitation and 530 nm emission band pass filters (ID-FISH, Milpitas, CA, USA), as described [ 9 ].…”

“…However this method cannot identify Babesia parasites at the species level. Alternatively, Babesia parasite nucleic acid is detected by qPCR on blood samples [ 3 , 4 ] and the detection of ribosomal RNA within infected red blood cells (iRBCs) by fluorescence in situ hybridization (FISH) [ 9 ]. Several qPCR tests have been developed for B. microti and are reported to detect <10 parasites per μL of blood [ 10 , 11 ].…”

“…Although a qPCR test for B. duncani has been recently developed [ 14 ], it is not yet in common use. The detection of B. duncani and B. microti in blood with the Babesia genus-specific FISH has been estimated to have a limit of detection of 58 parasites per μL of blood and provides highly specific laboratory confirmation of babesiosis [ 9 ]. Despite a lower sensitivity of detection, the Babesia genus-specific FISH test has several advantages, including lower resource and shorter time requirements, over qPCR tests, and these have been previously discussed in detail [ 9 ].…”

“…The detection of B. duncani and B. microti in blood with the Babesia genus-specific FISH has been estimated to have a limit of detection of 58 parasites per μL of blood and provides highly specific laboratory confirmation of babesiosis [ 9 ]. Despite a lower sensitivity of detection, the Babesia genus-specific FISH test has several advantages, including lower resource and shorter time requirements, over qPCR tests, and these have been previously discussed in detail [ 9 ]. However, Babesia parasite concentrations in peripheral blood can be low very early in an infection and during chronic low grade infections where parasites may be sequestered by binding to capillary endothelia in internal organs.…”

“…It is in this context that we report findings from the concomitant use of the recently developed Babesia genus-specific FISH test for detecting Babesia parasites in the blood [ 9 ] and two indirect IFA tests for separately detecting IgM and IgG antibodies against both B. duncani and B. microti for the laboratory diagnosis of babesiosis. The tests were performed on clinical blood samples received from the USA, Australia and Europe for routine testing for babesiosis and had the added advantage of being able to determine the relative prevalence of B. duncani and B. microti .…”