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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Malaria can be transmitted by the inoculation of blood from an infected donor.3 Transfusion-induced Plasmodium falciparum malaria has increased in recent years, probably because the parasite has become increasingly resistant to many drugs.4 Large numbers of parasites can be transmitted through this route of infection. Most patients in need of a blood transfusion are usually weakened by severe disease. Malaria behaves very aggressively in such patients with a higher risk of complications and fatalities.5
Infections with P. falciparum usually decrease within one year, those with P. vivax and P. ovale usually decrease within three years, but those with P. malariae can persist for as long as 50 years. Plasmodium vivax and P. falciparum are the most common species that cause malaria. Plasmodium malariae is also important because of its chronicity and the difficulty in detecting it.3
During the first third of the 20th century, syphilis was a serious medical problem. However, with the introduction of blood banks, syphilis transmission by blood has become a rarity and does not constitute a serious problem because the spirochetes do not survive longer than four days at refrigerator temperature.6 Transmission of malaria through blood transfusions is a greater threat7 because of the capability of human plasmodia to survive in stored blood and even in frozen blood.8
In Sudan, the rate of infection with malaria parasites among blood donors has been estimated by polymerase chain reaction (PCR) to be 21%. This infectivity rate is considerable and many Sudanese patients are at a higher risk of transfusional malaria complications and fatalities.9 This risk is worsened by the fact that the elimination of parasites from blood in vivo, before donation, takes at least 48 hours.10 Therefore, the practical difficulties of this procedure and its unreliability have been reported.2
Systemic screening of possible donors is not a practical solution in malaria-endemic countries because parasitemia in blood donors is often too low to be detected by microscopy or antigen-detection techniques.2 Even when advanced techniques are used, several thousand parasites might be present in one unit of blood and still remain undetected.11 Furthermore, it is neither ethical nor desirable to transfuse infected blood into weak, ill patients because this undoubtedly aggravates their condition. Malaria caused by blood transfusion may also be difficult to control even when optimal doses of antimalarial drugs are used.
These facts necessitate the need for in vitro processing of donor blood that could provide fast and reliable results. Thus, we tested the effect of various concentrations of SP when added to donor blood. These concentrations of SP in a unit of transfused blood are much lower than those given to donors or patients. Research activities for assessment of antimalarial resistance conducted by Department of Biochemistry at the University of Khartoum concluded that chloroquine resistance is increasing, quinine resistance is emerging, but that SP is still fully effective (Khalil IF, unpublished data). The purpose of this study was to determine the lethal dose of SP that eliminates malaria parasites in vitro and the effects of this dose on stored blood.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sample collection took place between October 2002 and January 2004. All individuals who donated blood in Khartoum state hospitals during the study period were included in the studied population if they satisfied the following criteria: presence of malaria parasites in blood films; parasitemia between 1,000 and 80,000 parasites/µL (only asexual stages were considered); growth of parasites on the first day when cultured; and donors did not take quinine in the past 7 days, chloroquine in the past 28 days, and SP in the past 14 days.
One unit (200 mL containing 28 mL of citrate phosphate dextron adenine-1) of malaria parasite–infected donor blood was collected and divided into four equal samples (50 mL) using a blood bank mixer (Biomixer 323; Abelko Innovation, Luleå, Sweden). Three of the samples received the appropriate drug dilution and the other received no drug (control). All blood specimens were tested for parasite culture, platelets count, total leukocyte count, packed cell volume (PCV), percent lysis, osmotic fragility, prothrombin time, activated partial thromboplastin time, serum sodium and potassium levels simultaneously on the day of collection. The blood was then stored in the blood bank refrigerator (4–6°C) and tested after 24 and 48 hours by the same laboratory procedures.
Microscopic identification of malaria parasites was performed as described by Cheesbrough.11 The absolute number of parasites per microliter was determined in thick blood films by counting the number of parasites against 200 white blood cells, multiplying by the total leukocyte count, and dividing by 200. In vitro cultivation of erythrocytic stages of P. falciparum to assess parasite response to SP and confirm the viability or the death of the parasites was conducted using RPMI 1640 medium (Invitrogen, Carlsbad, CA). The minimum inhibitory concentration was determined by computerized probit/log dose response analysis (SPSS, Inc., Chicago, IL). Formation of schizonts at a concentration of 300/
3.75 pmol of SP confirmed resistance.
For counting platelets and white blood cells, blood samples were diluted 1:20 in 1% ammonium oxalate and 2% glacial acetic acid consecutively as described by Cheesbrough.11 Partial thromboplastin time, a screening test for the intrinsic clotting system, i.e., factors XII, XI, IX, VIII, X, and V, prothrombin, and fibrinogen, and prothrombin time, a screening test for the extrinsic clotting system, i.e., factors VII, X, and V, prothrombin, and fibrinogen, were conducted. These tests were conducted as described by Dacie and Lewis12 using a commercial reagent (DiaMed Company, Turnhout, Belgium). The osmotic fragility test, a measure of the surface area/volume ratio of erythrocytes and the effect of various drug doses on erythrocytes, was conducted as described by Dacie and Lewis.12 Packed cell volume, the proportion of whole blood occupied by erythrocytes, was measured by the micro-hematocrit method described by Dacie and Lewis.12
Percentage lysis is an indicator of survival of erythrocytes during the storage period. Five milliliters of each blood sample just after collection were centrifuged for 5 minutes at 12,000 x g. The supernatant was stored at 4°C and used as a control. Five hundred microliters of the centrifuged erythrocytes were diluted in 5 mL of distilled water, stored at 4°C, and used as standard. Both solutions were used in the determination of percentage lysis for the same sample throughout the storage period. One milliliter of each blood sample after 24 and 48 hours was also centrifuged for 5 minutes at 12,000 x g and the supernatant (plasma) of each was tested for hemolysis at a wavelength of 540 nm. Percentage lysis was calculated by dividing the optical density of the tested supernatant by the optical density of the standard and multiplying by 100.
Serum levels of potassium and sodium were measured by the flame photometry using a 410 flame photometer (Sherwood Scientific, Cambridge, United Kingdom).
Data were analyzed using SPSS software (SPSS, Inc.). Non-parametric tests were used for abnormally distributed data.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1,000 in 63 (22.7%) specimens. Of these, 30 (10.8%) samples were resistant to chloroquine and three (1.08%) additional samples showed no growth on the first day when cultured. Thus, the number of blood samples was reduced according to the above-mentioned parameters to 30. All of the accepted blood bags were from asymptomatic male donors between 25 and 35 years of age.
The reduction in malaria parasites when different concentrations of SP were added to donor blood and stored for 48 hours is shown in Table 1
. The control sample of donor blood (without SP) showed stable numbers of parasites even after storage for 48 hours. Parasite counts decreased with increasing concentrations of SP and a longer storage period. Parasites were not detectable after 24 hours in blood samples with higher concentrations of SP (200 and 300 µg/L) and after 48 hours in samples containing the minimum concentration (100 µg/L) of SP.
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. These doses were high when donor blood was stored for 24 hours and lower in blood samples stored for a longer time. Lethal doses also gradually increased when a large number of malaria parasites (99%) were killed (LD99) and decreased when lower numbers of the parasites (50%) were killed (LD50).
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). They reached a peak in samples with SP concentrations of 200 and 300 µg/L. The minimum increase was observed with the minimum concentration of SP. Although there was a statistically significant difference between the results among different blood samples stored for the same time period (P < 0.05), the upper levels were within the normal range.
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. The mean osmotic fragility values reached a peak mainly after storage for 48 hours. No statistically significant difference was observed between the results of the control and the drug-treated blood samples when stored for 24 hours except in samples with a final SP concentration of 300 µg/L. When blood samples were stored for 48 hours, no difference was observed between drug-treated blood samples and the control in samples containing 100 µg/L of SP. Differences were observed in samples containing 200 and 300 µg/L of SP; however, the upper levels were within the normal range.
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). Samples with a final concentration of 200 or 300 µg/L of SP had significantly different PCVs compared with the control when stored for 24 or 48 hours. However, this statistical difference was within the normal range.
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). The number of platelets correlated with the dose concentration adverse to white blood cell counts. No statistically significant difference was observed between the results of the control and drug-treated blood samples.
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). A statistically significant difference was observed between the control and blood samples containing the highest dose of SP (300 µg/L), but no statistically significant difference was observed between the control and drug-treated blood samples. The amount of lysis observed in these blood samples was insignificant.
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). A statistically significant difference was observed between the control and blood samples at all three drug doses. Serum potassium levels also increased at high drug concentrations and at longer storage times. After storage for 24 hours, a statistically significant difference (P < 0.05) was observed between the results of the control and blood samples containing 100 and 200 µg/L of SP. Conversely, after 48 hours, no association was observed between results of the control and all blood samples containing 300 µg/L of SP. The upper levels of both electrolytes were both within the normal range for stored blood.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Direct microscopic examination of donor blood is of little value. Such a time-consuming procedure is obviously impractical, especially with regard to asymptomatic blood donors who often have low parasite densities at the submicroscopic level.13 Detection of malaria antibodies provides evidence of an immune response to current or past infection. However, these test results may remain positive for more than 10 years after the parasitemia has resolved. Therefore, detection of malaria antibodies to screen blood donations would result in the exclusion of otherwise healthy persons.14
Although the PCR has an increased sensitivity compared with blood film examination, several thousand malaria parasites might be present in a unit of blood (450 mL) and still not be detected.11 This number of parasites is much larger compared with the 12–20 sporozoites delivered by the mosquito bite.15 This indicates the ineffectiveness of testing blood donors to eliminate the risk of malaria transmission by blood transfusion, which is consistent with the failure of the screening system used by blood banks in the United States to prevent the occurrence of such cases.7
In the present study, the number of parasites and parasites densities in blood samples treated with three doses of SP showed a significant difference when stored for 24 hours. Nevertheless, they were insignificantly different when blood samples were stored for 48 hours. This is likely due to effectiveness of the different concentrations of SP, which was reflected by the effect of even low concentrations of this drug.
The lethal dose of SP (179.65 µg/L) was greater than that reported by Bruce-Chwatt in 1985 (10–100 µg/L). This increase is probably due to the behavior of the parasite towards the drug over the previous 19 years, which has resulted in adaptation of the parasite to become relatively resistant. Also, the difference in the strains may explain this increase.
The effect of SP on all constituents of stored blood was assessed to ensure whether the doses of drugs were safe. The mean prothrombin time and partial thromboplastin time, which are indicators of the clotting system, increased in samples containing higher drug concentrations. However, this increase also showed a correlation with the time of storage. Even at the higher drug dose, the increase in these times was acceptable. Thus, the lethal dose of the drug does significantly affect plasma coagulation factors.
The osmotic fragility test, which measures the functions and integrity of the erythrocyte membrane, was used to measure the effect of SP on the malaria parasite and the erythrocyte membrane. Mean osmotic fragility values were significantly correlated with the storage time; they increased when blood samples were stored for 48 hours compared with those stored for 24 hours. However, lethal doses of the drug had no effects on the flexibility of the erythrocyte membrane.
The hematocrit is an indication of shrinking or swelling of red blood cells. In stored blood, hematocrit values are decreased because of dilution by the anticoagulant solution. Some investigators have described a relative increase in hematocrit values of stored blood with an increase in storage time due to the entry of plasma fluids into the erythrocytes during storage.6 In the present study, PCV values decreased proportionally with the length of the storage period. However, SP did not significantly affect these values. The red blood cells that hemolyzed during storage may explain the decrease in PCV in the present study.
We also studied the effect of SP on platelet counts. This study showed a significant decrease in platelet counts that correlated with increasing concentration of SP. However, the effect of lethal doses of SP on platelet counts appears to be acceptable; counts were within the reference values for stored blood.
Percent lysis in stored blood reflects the unfavorable effect of storage or applied drugs on the viability of erythrocytes. This value in the present study increased in proportional to the concentrations of the drug as well as the storage period. Although the highest value of lysis was 0.22%, it was low compared with that reported in normal stored blood (1%) by Mollison.6
Storage of donor blood results in an increase in both sodium and potassium levels, which is likely to be due to inactivation of active transport of these electrolytes across the red blood cell membrane. Blood samples showed a poor correlation between sodium levels and drug concentration, despite a good correlation with the storage period. However, these findings were not observed for potassium levels. They did not show a correlation with the drug concentration, but did show a correlation with the duration of storage. However, the increase in the potassium level was acceptable when blood samples were treated with the lethal dose of SP.
We have shown that in vitro treatment of donor blood with SP is simple and applicable because the concentrations of SP used can be safely added to the constituents of stored blood (anticoagulant and preservatives) without incompatible interactions. Moreover, this process is inexpensive because one ampule of SP can be used for 250 blood units (bags). We recommend that SP be added to stored blood to give a final concentration of 180 µg/L. Future studies are recommended to select highly effective and safe antimalarial drugs based on patterns of resistance and side effects and to assess the safety of this process.
Received April 1, 2005. Accepted for publication May 26, 2005.
* Address correspondence to Mohamed S. M. Ali, Department of Haematology, Faculty of Medical Laboratory Sciences, Al Neelain University, P.O. Box 12702, Khartoum, Sudan. E-mail: mohdaru{at}hotmail.com 
Authors’ addresses: Mohamed S. M. Ali, Department of Haematology, Faculty of Medical Laboratory Sciences, Al Neelain University, Khartoum, Sudan, E-mail: mohdaru{at}hotmail.com. Abdul G. M. Y. Kadaru, Faculty of Medicine, University of Khartoum, Khartoum, Sudan.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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M. S. M. Ali Letters to the editor. Am J Trop Med Hyg, May 1, 2006; 74(5): 705 - 705. [Full Text] [PDF]
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