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CLINICAL REVIEW: Christopher J M Whitty, Mark Rowland, Frances Sanderson, and Theonest K Mutabingwa Science, medicine, and the future: Malaria BMJ 2002; 325: 1221-1224 [Full text]

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Malaria diagnosis, a neglected area of malaria research

Chansuda Wongsrichanalai   (9 December 2002)

Treatment or Protection?

Jonathan L. Cumblidge   (11 December 2002)

Indole-3-Carbinol: a novel anti-parasitic treament?

Daniel R McGrath, Hamid Frydoonfar, Allan D. Spigelman   (21 January 2003)

Malaria diagnosis, a neglected area of malaria research 9 December 2002
Chansuda Wongsrichanalai, Epidemiologist 130 Sub Street, Bangkok 10500, Thailand Send response to journal: Re: Malaria diagnosis, a neglected area of malaria research	While preventing transmission by mosquitoes, vaccination, and drug treatment are the three rapidly-developing areas of malaria research, research with immediate practical applications in endemic countries is moving slowly (1). A good example of the latter is the neglected research on malaria diagnosis. After a century of its existence, Giemsa microscopy, the current standard of malaria diagnosis, has not yet effectively reached a vast area where it is most needed. Field microscopy in many remote endemic areas is known to be of poor quality or unavailable and clinical diagnosis continues to be the mainstay (2). The availability of malaria dipsticks in the last decade gave a fault hope for replacing microscopy and undermined capability building in microscopic diagnosis. Despite its advantages, dipstick today is known for several limitations: detection threshold is at least a few hundred parasites per ml blood, inability to distinguish malaria species aside from P. falciparum, its unit price not affordable by most malaria control programs, etc. (2). Worse yet, recent problems associated with irregular dipstick qualities question the readiness to deploy malaria dipsticks to endemic areas (3). Funding for diagnostic research is scarce compared to the three major areas mentioned by Whitty et al.(1). No additional rapid diagnostic assay has been introduced since the establishment of HRP2 and pLDH as assay targets in the 1990s. Accurate malaria diagnosis is a foundation for malaria control. Without it, the impact of control measures cannot be reliably assessed; outcomes of therapeutic trials may be flawed (4). According to Whitty et al., two areas of scientific research are undergoing in regard to malaria treatment: finding novel drugs and determining a means for effective use of existing drugs (1). However, parallel efforts to improve laboratory diagnosis of malaria and to bring accurate diagnosis into practice in the poor countries where malaria remains a major public health burden are also essential. If antimalarial drugs are still prescribed largely based on clinical diagnosis, their lifespan will be short and we will forever be compelled toward the unfinished search of new drugs. Unless funding agencies begin to recognize the importance of malaria diagnosis, its improvement is unlikely to happen soon. References: 1. Whitty CJ, Rowland M, Sanderson F, Mutabingwa TK. Science, medicine, and the future: Malaria. BMJ 2002; 325: 1221-4. 2. WHO. New perspectives, malaria diagnosis. Report of a joint WHO/USAID informal consultation, 25-27 October 1999. Geneva, Switzerland: World Health Organization, 2000. (WHO/CDS/RBM/2000.14 WHO/MAL/2000.1091). 3. Wongsrichanalai C, Miller RS. Malaria rapid tests: a public health perspective. Lancet 2002; 359: 1781. 4. Ohrt C, Purnomo, Sutamihardja MA, Tang D, Kain KC. Impact of microscopy error on estimates of protective efficacy in malaria-prevention trials. J Infect Dis 2002; 186: 540-6. Competing interests:   None declared
Treatment or Protection? 11 December 2002
Jonathan L. Cumblidge, Medical Scientist PathCentre, Locked Bag 2009, Nedlands, W.A. 6909, Australia Send response to journal: Re: Treatment or Protection?	Some of my work involves diagnosis of malaria in the laboratory by reviewing blood films thick and thin (the latter at two pH's), and by using the ICT monoclonal "card", which is supposed to be highly sensitive and specific for falciparum and vivax. I have worked in a hospital laboratory in Flores, Indonesia, where diagnosis was made using thick and thin films. Despite teaching from medical scientists from the U.K., the Indonesian laboratory in question still resorted to using recycled slides and both films stained using only the technique used for all other routine blood films. The hospital grounds were occasionally sprayed using a combination of DDT and Lindane, but mosquitoes still persisted within days due to the proliferation of pools of water all over the hospital. Only the patients who could afford it had fly screens on their windows (but no bed nets), and the poor patients had no glass in the windows of their wards, let alone fly screens! There was therefore a vast source of blood meals for the mosquitoes. Most of the population of the island had grown up developing a mild immunity to malaria, so when adults contracted it they did not become bed- ridden as foreign tourists would (including myself!). As a consequence there was never a compulsion amongst Floresians to combat the disease. The insecticide program was merely a show put on by the Indonesian government. Most of the locals I got to know prevented getting bitten by mosquitoes simply by showering everyday before dusk and wearing fresh clean clothes. I found that mosquitoes responded most voraciously to sweaty "Westerners" whose heat and smell are so attractive! It is widely accepted that treatment for malaria should only begin after diagnostic confirmation. As most of the population of Flores would simply tolerate the disease, treatment is rarely sought, therefore the easy-to-use ICT cards are pointless. Local beliefs about protection from getting bitten are unlikely to extend beyond good personal hygiene, burning anti-mosquitoes "coils" (which are largely ineffective) and the use of insect repellent cream (one brand of which I found to be very effective and widely available). Eradication of mosquitoes on Flores seems to be an impossible task, due to ineffective measures (in scope and thoroughness), apathy and a willingness of the population simply to put up with malaria as a fact of life and death, especially when bigger issues such as poverty and malnutrition hamper the quality of life on the island. If my view of how malaria lives on in Flores is a snapshot of all the World's tropical zones, then I think developing vaccines and bed-nets still has a long, long way to go. Competing interests:   None declared
Indole-3-Carbinol: a novel anti-parasitic treament? 21 January 2003
Daniel R McGrath, Lecturer, Surgical Science University of Newcastle, NSW 2308, Australia, Hamid Frydoonfar, Allan D. Spigelman Send response to journal: Re: Indole-3-Carbinol: a novel anti-parasitic treament?	Many established and emerging infections which afflict mankind, particularly large parasitic organisms, continue to elude effective anti microbial therapy. These organisms cause widespread morbidity and mortality in developing nations 1. The plant extract Indole-3-carbinol (I3C), a metabolite of the glucosinolate Glucobrassicin, is found almost exclusively in the Brassica genus of vegetables which includes broccoli and cabbage. This compound has found use in the treatment of various conditions as diverse as cervical intra-epithelial neoplasia 2 and fibromyalgia. Metabolites of other glucosinolates are known to have anti-bacterial, anti-fungal and anti- protozoal activities 3. With the advent of increasing drug resistance alternative drug treatments are being sought. One such group, the artemisin group (derived from a Chinese herbal drug), in combination with existing treatments has shown significant efficacy 1. During the course of other investigations, some of our cells became infected with a Raphidiophrys species and it was observed that growth of this species was inhibited in those wells containing I3C. We hypothesised that the presence of I3C was inhibiting parasitic growth. A literature search revealed no previous reports of I3C and parasitic growth inhibition. We have tested our hypothesis by exposing the parasite Plasmodium falciparum (FCQ27) to varying concentrations of I3C and measuring the effect on growth inhibition. Red blood cells infected with Plasmodium falciparum (strain FCQ-27) were exposed in 96 well culture plates for 40 hours to varying concentrations (3.125 – 100 mmol) of I3C. The Control group contained the same culture media without I3C. Both groups were incubated in a gas mixture of 5% O2, 5% CO2 and 90% N2 at 37°C for 24h. Plasmodium falciparum growth was assessed by measuring the incorporation of 3.7KBq of [G-3H] hypoxanthine, a radio-labelled nucleic acid precursor. On addition of [G-3H] hypoxanthine, incubation continued under the same conditions for a further 18-20 hours. Cells were then harvested and scintillation counting employed to measure cell-associated radioactivity. The lack of hypoxanthine incorporation implied toxicity. Results were confirmed by estimation of the percentage of parasitaemia in all cells determined by the microscopic examination of a Giemsa stained blood smear obtained from each well. Results were based on the mean value of triplicate experiments. Statistical analysis was carried out using the Mann–Whitney U-test (MWU). I3C inhibited the growth of Plasmodium falciparum (Figure 1). The LD50 was found to be approximately 0.65mmol. This was confirmed by assessment of parasitaemia. Parasitaemia was found in 6-8% of the Control group and in less than 0.1% of the Test group at this level (p=0.005). The growth of Giardia trophozoites and the protozoa Raphidiophrys was also inhibited by I3C (Data not shown). Figure 1 Plasmodium falciparum malaria is the most frequent and problematic form of malaria. The greatest burden of this strain of malaria is carried by those living in sub-Saharan Africa. Falciparum malaria is less common in South America, the Indian subcontinent and Southeast Asia 4. In particular areas of Southeast Asia, cruciferous vegetables form a large proportion of the staple diet 5. Could this greater consumption of cruciferous vegetables account for the lesser prevalence of Falciparum malaria in such areas? This has yet to be determined; however the consumption of cruciferous vegetables may be a potential treatment or synergistic agent which is both inexpensive and potentially widely available fulfilling the requirements of new anti-malarials 1. The Plasmodium falciparum strain is very drug resistant. Chloroquine is still the mainstay of treatment in some areas despite failure rates of up to 70% 4. Glutathione and Glutathione S-Transferase are known to play an integral role in the development of chloroquine resistance. I3C is known to interact with Glutathione and stimulate Glutathione S-Transferase in mammalian cells 3. Despite the preliminary nature of this work these experiments suggest that I3C is worthy of further investigation as an anti-parasitic agent. Our work was carried out on one strain of Plasmodium falciparum and future studies on different strains would be useful. The effect of I3C on Giardia, Raphidiophrys and Plasmodium falciparum suggest that the role of I3C might be extended to cover most of the organisms in the protist family. I3C has been administered to humans at a dose of 400 mg/day for a period of three months without side effects 2. As this dose is within the effective range of inhibition (as determined in-vitro) a pilot clinical study of patients with uncomplicated Plasmodium falciparum is feasible. References 1. Whitty CJM, Rowland M, Sanderson F, Mutabingwa TK. Science, medicine, and the future: Malaria. BMJ 2002;325(7374):1221-1224. 2. Bell MC, Crowley-Nowick P, Bradlow HL, Sepkovic DW, Schmidt-Grimminger D, Howell P, et al. Placebo-controlled trial of indole-3-carbinol in the treatment of CIN. Gynecologic Oncology. 2000;78(2):123-9. 3. Talalay P, Fahey JW. Phytochemicals from cruciferous plants protect against cancer by modulating carcinogen metabolism. Journal of Nutrition. 2001;131(11 Suppl):3027S-33S. 4. Winstanley PA. Chemotherapy for falciparum malaria: the armoury, the problems and the prospects. Parasitology Today. 2000;16(4):146-53. 5. Seow A, Shi CY, Chung FL, Jiao D, Hankin JH, Lee HP, et al. Urinary total isothiocyanate (ITC) in a population-based sample of middle-aged and older Chinese in Singapore: relationship with dietary total ITC and glutathione S-transferase M1/T1/P1 genotypes. Cancer Epidemiology Biomarkers and Prevention 1998;7(9):775-81. Figure 1 Figure 1 Legend Inhibitory effect of in-vitro Indole-3-carbinol on P. falciparum, strain FCQ-27, detected by scintillation counting (p = 0.005). Competing interests: None declared