Plasmodium DNA Fluoresces With Berberine A Novel Approach for ...

Microbiology and Infectious Disease / PLASMODIUM DNA FLUORESCES WITH BERBERINE

Plasmodium DNA Fluoresces With Berberine A Novel Approach for Diagnosis of Malarial Parasites Sameer S. Talwalkar, MD,1 A.B. Vaidya, MD, PhD,2 Chhaya Godse, PhD,2 Akhil Vaidya, PhD,3 and Rama Vaidya, MD, PhD2 Key Words: Malaria; Plasmodium vivax; Plasmodium falciparum; Berberine; Isoquinoline alkaloids DOI: 10.1309/CG7037YYPBUBV703

Abstract The key to reducing mortality and morbidity associated with malaria is rapid diagnosis and early, effective therapy. Berberine, a plant alkaloid, has been used for fluorescent staining of the Y chromosome. We evaluated whether berberine can be used for staining of malarial parasites in 40 selected peripheral blood smears from patients with clinical symptoms of malaria; smears were evaluated with OptiMal (DiaMed, Miami, FL) and Giemsa stain. Twenty were positive with both OptiMal and Giemsa (Plasmodium vivax, 14; Plasmodium falciparum, 6); 10 were negative with both. The remainder were positive by OptiMal but negative by Giemsa and, therefore, were classified as equivocal. All slides were processed simultaneously, stained with berberine, and read under a fluorescent microscope. P vivax and P falciparum DNA fluoresced with berberine. The positives and negatives by berberine concurred with the Giemsa staining. Of the 10 equivocal smears, 5 were confirmed positive by berberine. Gametocytes were easily identifiable. This test has high sensitivity and high positive predictive value and, once standardized, can be used as a potential screening and diagnostic tool.

Malaria has assumed alarming status in the developing world. Annually, 500 million people are infected with malaria, with more than 2.7 million deaths.1 Owing to the high mortality associated with this infection, new approaches for its diagnosis and management and drug development are essential. Berberine, which belongs to the isoquinoline group of alkaloids, is unique because of its ability to intercalate and form a stable complex with DNA. We explored this property of the alkaloid in the present study. The aim was to evaluate whether berberine stains the plasmodial DNA so that it could be used as an effective tool for rapid screening and early diagnosis of malaria.

Materials and Methods Berberine Preparation The materials required for the preparation of berberine stain included the following chemicals: (1) ethyl alcohol in serial dilutions of 80%, 70%, and 50%; (2) phosphate buffer, pH 7.0, prepared by addition of 0.1 mol/L of disodium hydrogen phosphate, 4.73 g, and potassium dihydrogen phosphate, 4.5 g, to 1 L of distilled water; and (3) calcium chloride prepared by the addition of 50 g of calcium chloride amorphous powder to 500 mL of distilled water. Berberine solution (pH 7.4) was prepared by the addition of 1.75 g of berberine powder to 500 mL of distilled water. This solution then was heated at 40°C to dissolve the berberine completely. Selection of Cases The 40 slides used for staining were all thin, peripheral blood smears obtained from patients with clinical symptoms of

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Microbiology and Infectious Disease / ORIGINAL ARTICLE

malaria. The cases selected for the study were based on the results of the OptiMal test (DiaMed, Miami, FL) followed by subsequent confirmation by Giemsa staining of the peripheral smears. All cases occurred before the initiation of antimalarial therapy. Of the 40 cases, 20 were positive with both OptiMal and Giemsa (Plasmodium vivax, 14; Plasmodium falciparum, 6), whereas 10 were negative with both. The 10 equivocal cases were positive with OptiMal, but Giemsa staining did not reveal ring forms or gametocytes. Instead, only pink dots were seen within the RBCs. Therefore, these 10 cases were classified as equivocal. Slides from all cases were fixed in methanol, air dried, and preserved at room temperature in a fluorescent slide fixative consisting of ethanol and ether before staining with berberine.

cases negative by Giemsa were confirmed as negative by berberine. Of the 10 equivocal cases, 5 were confirmed to be positive with berberine by the presence of fluorescing rings, whereas the remaining 5 showed occasional fluorescing green dots within the RBCs. In the negative slides, no such dots were seen. Species differentiation was difficult by this technique; however, in 2 of 6 P falciparum cases, identification of the species was possible based on the morphologic features of the ring. Gametocytes were easily identifiable based on their morphologic features and bright fluorescence ❚Image 2❚ and ❚Image 3❚. The cases that were positive with both OptiMal and Giemsa had moderate to high degrees of parasitemia ❚Image 4❚, ❚Image 5❚, and ❚Image 6❚. In the equivocal cases, it was difficult

Staining Method Each slide to be stained first was rinsed in serial dilutions of ethanol (from 80% to 50%) for 10 seconds each. The slides then were rinsed with distilled water followed by immersion in berberine solution for 3 minutes. At the end of 3 minutes, the slides were immersed directly into phosphate buffer for 1 minute so as to remove the excess stain. Finally, the slides were placed in calcium chloride for 1 minute to enhance the differentiation between RNA and DNA. Immediately following this step, the slides were mounted using Vectashield (Vector Labs, Burlingame, CA), a fluorescent mounting medium, while they were still wet and then sealed using DPX. In this way, all the 40 slides were sealed, placed in a closed box to protect from fading due to exposure to light, and then placed in a refrigerator at 4°C to 6°C for 48 hours before viewing. Evaluation of Smears All slides stained with Giemsa and berberine were examined independently by 2 pathologists (S.S.T. and C.G.). Species identification and grading of Giemsa-stained smears was based on counting the number of parasites in 300 oil-immersion fields. Berberine-stained slides were observed under the confocal fluorescent microscope using the blue laser at a wavelength of 580 λ for excitation. The images were captured under the microscope and transferred to the Confocal Assistant, specialized software that scans the images and takes sections through different planes of thickness. Thus, a projection image was obtained, which was modified for color, brightness, and contrast using Adobe Photoshop, version 6.0 (Adobe, San Jose, CA), to facilitate printing. The confocal fluorescent microscope was used purely for research purposes to facilitate capturing high-quality images.

❚Image 1❚ Ring-formed trophozoite of Plasmodium vivax (berberine, ×1,000).

Results Smears containing both P vivax and P falciparum fluoresced with berberine. All 20 cases positive by Giemsa were confirmed as positive with berberine ❚Image 1❚, whereas all 10

❚Image 2❚ Gametocyte of Plasmodium falciparum (berberine, ×1,000).

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to estimate the level of parasitemia because there were no well-defined parasitic morphologic features as visualized with Giemsa staining. However, these cases were positive by OptiMal, and, on staining with berberine, 6 of 10 cases showed fluorescing ring forms ❚Image 7❚ and ❚Image 8❚.

The case fatalities due to malaria are increasing. The key to reducing mortality and morbidity is rapid diagnosis and early, effective therapy. The “gold standard” for diagnosis of malaria has been smear microscopy. This may be time-consuming

and requires trained and experienced individuals to examine the blood smears, especially with low parasitemia. As a consequence, rapid diagnostic tests, which include OptiMal, ParaSight F (Becton Dickinson, Franklin Lakes, NJ), and the ICT card test (AMRAD, Sydney, Australia), are now commercially available and are used widely for the rapid screening of suspected cases and also for the follow-up of the treated patients.2 However, these tests are relatively expensive and a constraint for use in the endemic areas. In addition, the card test cannot distinguish between the antigen of a viable and nonviable parasite, its sensitivity decreases at lower levels of parasitemia,3 and sometimes even very high levels of parasitemia (>1,000 parasites per microliter) give false-negative results.4

❚Image 3❚ Gametocyte of Plasmodium falciparum (berberine, ×400).

❚Image 4❚ Two intraerythrocytic ring-formed trophozoites of Plasmodium vivax (berberine, ×1,000).

❚Image 5❚ Intraerythrocytic schizonts of Plasmodium vivax before their release with lysis of the RBC (berberine, ×400).

❚Image 6❚ Intraerythrocytic schizonts of Plasmodium vivax (berberine, ×400).

Discussion

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The quantitative buffy coat method (Becton Dickinson) is more accurate and sensitive; it is a capillary fluorescence method.5 Acridine orange is used in this method and has been documented as a fluorescent stain for the detection of malarial parasites.6 The limitations of this method are the cost per test and the initial investment for equipment such as a microhematocrit centrifuge and a UV light adapter. Berberine, which belongs to the isoquinoline group of alkaloids, was isolated in 1926. Berberine has numerous interesting pharmacologic effects.7 Berberine and related alkaloids are widely distributed in numerous plants such as Berberidaceae, Menispermaceae, Ranunculaceae, and Rutaceae.8 Some of these alkaloids have antimicrobial activity against a variety of microorganisms, including fungi and protozoa. Several publications exist on the antibacterial activities of berberine against Vibrio,9 Bordetella, Salmonella, and Escherichia organisms.10-12 What is most fascinating are cytotoxic13 and antineoplastic effects14 of berberine against tumor cells. Limited studies have been carried out to explore this potential, but there is a good reason to believe that these activities are due to the ability of berberine to readily intercalate and form a stable complex with DNA.15 Berberine hydrochloride has been used as a fluorescent dye for staining the Y chromosome, and it has been documented that it brightly fluoresces the long arm of the Y chromosome in the metaphase and the Y chromatin in interface nuclei.16 As a follow-up of this work, we used this alkaloid to stain malarial parasite DNA in peripheral blood smears. Our findings demonstrated that berberine binds effectively to plasmodial DNA. This screening test has high sensitivity and a high positive predictive value. It is simple and

❚Image 7❚ Equivocal case, confirmed negative with berberine (×400).

cost-effective to set up in a laboratory or a field setting. Because the DNA binding is nonspecific, berberine potentially could be used for identification of other blood protozoa, especially Babesia organisms. The next step would be to standardize this test with respect to the minimum concentration of berberine required for fluorescence. In addition, the affinity of berberine to bind to nucleic acids needs to be studied. Once standardized, it can be used as a potential screening and diagnostic tool for early detection, monitoring of drug treatment, and follow-up of patients with malaria. From the 1Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, KY; 2Bhavan’s SPARC Research Center, Mumbai, India; and 3Department of Microbiology and Molecular Genetics, Drexel University, Philadelphia, PA. Address reprint requests to Dr Talwalkar: Dept of Pathology and Laboratory Medicine, University of Louisville Hospital, 530 S Jackson St, Louisville, KY 40220. The abstract of this article was selected as a finalist for oral presentation at the Sheard Sanford competition at the ASCP Annual Meeting, October 2004, and subsequently was published (Am J Clin Pathol. 2004;122:630). Acknowledgment: We thank Shankar Jha, PhD, and Vidita Vaidya, PhD, for their wholehearted support and technical help in this project.

References 1. World Health Organization. Management of Severe Malaria: A Practical Handbook. 2nd ed. Geneva, Switzerland: World Health Organization; 2000:239-242. 2. Moody A, Manser D. Laboratory practices for the diagnosis of malaria. Clin Lab Int CLI. September 2001:12-14.

❚Image 8❚ Equivocal case, confirmed positive for intraerythrocytic ring-formed trophozoites of Plasmodium vivax with berberine (×400).

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