The use of fluorescence enhancement to improve the microscopic diagnosis of falciparum malaria

Abstract

Background: Giemsa staining of thick blood smears remains the "gold standard" for detecting malaria. However, this method is not very good for diagnosing low-level infections. A method for the simultaneous staining of Plasmodium-parasitized culture and blood smears for both bright field and fluorescence was developed and its ability to improve detection efficiency tested.

Methods: A total of 22 nucleic acid-specific fluorescent dyes were tested for their ability to provide easily observable staining of Plasmodium falciparum-parasitized red blood cells following Giemsa staining.

Results: Of the 14 dyes that demonstrated intense fluorescence staining, only SYBR Green 1, YOYO-1 and ethidum homodimer-2 could be detected using fluorescent microscopy, when cells were first stained with Giemsa. Giemsa staining was not effective when applied after the fluorescent dyes. SYBR Green 1 provided the best staining in the presence of Giemsa, as a very high percentage of the parasitized cells were simultaneously stained. When blood films were screened using fluorescence microscopy the parasites were more readily detectable due to the sharp contrast between the dark background and the specific, bright fluorescence produced by the parasites.

Conclusion: The dual staining method reported here allows fluorescence staining, which enhances the reader's ability to detect parasites under low parasitaemia conditions, coupled with the ability to examine the same cell under bright field conditions to detect the characteristic morphology of Plasmodium species that is observed with Giemsa staining.

Figure 1

Effect of EMA. P. falciparum-parasitized red blood cells were stained with photoactivated ethidium monoazide (EMA) alone or with photoactivated EMA followed by Giemsa. The images on the top are viewed under light microscopy and the lower images are the same field viewed using fluorescent microscopy.

Figure 2

Staining of P. falciparum-infected red blood cells. Panel A depicts two different fields of stained P. falciparum-parasitized red blood cells, both viewed using light microscopy. The left hand side was stained with Giemsa alone and the right hand side was stained with Giemsa followed by SYBR Green 1. Panel B depicts the same field of dual stained P. falciparum infected red blood cells viewed under light (left hand side) or fluorescence (right hand side).

Figure 3

Dual Giemsa and SYBR Green 1 staining of P. falciparum inoculated blood. The top panel represents blood films visualized under light microscopy and the lower panel represents the same field visualized using fluorescence microscopy.

Figure 4

LEDs can be used for detecting SYBR stained pRBCs. Fluorescence emission from pRBCs excited with a 5 mW Blue LED powered by 4 AA batteries. The pRBCs were stained with SYBR Green 1 and were then examined using a Zeiss Axiostar Plus microscope with an LED light source fitted in place of its mercury lamp. Photographs depict fluorescent (left) and white light (right) images of a mature parasite (upper) and a ring stage parasite (lower). Fluorescent intensity of the stained parasites was comparable to that observed with the HBO 50/AC high intensity light source supplied by the manufacturer.