Eradication of Transfusion-Induced Malaria by In vitro Processing

OBJECTIVE: To determine the lethal dose of chloroquine to be added to donors’ blood in vitro for eradication of transfusion-induced malaria and to study the unfavorable effects of this dose on the constituents of the stored blood. METHODS: A total of 4484 blood donors, recruited for this study, were screened for malaria parasites microscopically using Giemsa’ staining technique. Only 30 blood samples (200ml of blood each) satisfied the inclusion criteria of the study. Each of these blood samples was subdivided equally into four sub-samples to obtain 120 sub-samples. Three different concentrations of chloroquine were added to 90 specimens (30 samples represent each dose) while 30 specimens (control) were left without the drug. Blood specimens were then tested by parasitic, hematological and biochemical techniques on the day of collection and after 24 and 48 hours storage in blood bank refrigerator. RESULTS: The numbers of malaria parasites killed were proportional to chloroquine doses added to donors’ blood. No parasites were killed among the control donors’ blood samples. The determined lethal dose of chloroquine was safe to all constituents of the stored blood. CONCLUSION: For eradication of transfusion induced malaria by in vitro processing of donors blood, chloroquine is effective and safe drug. We recommend application of the optimal dose of chloroquine (626.1μg/L) to the components of the blood bags prior to phlebotomy.

All known species of malaria are able to survive for days and even weeks in blood stored at 4ºC 3 .Malignant tertian malaria (P.falciparum) has been transmitted by blood stored for 14 days 4 while benign quartan malaria (P.malariae) has been accidentally transmitted in blood previously stored for 5 days 3,5 .Consequently large numbers of parasites can be transmitted through this route where as, most patients in need of blood transfusion are already weakened by severe disease.Malaria thus behaves very aggressively in such patients 6 .In Sudan, the infection rate of the malaria parasite among blood donors was 6.5% using direct microscopy (61.9% sensitivity) and 21% using the PCR.This infectivity is T considerable and can lead to a higher risk of complications and fatality in recipients 7 .This risk is worsened by the practical difficulties of the elimination of parasites from donors' blood in vivo, before donation 1 .Also, systemic screening of suspected donors is not a practical solution to the problem in malarial endemic countries 1 .Even by using an advanced technique several thousand parasites might be present in one pint of blood and still pass undetected 8 .Moreover, it is neither ethical nor desirable to transfuse infected blood to weak, ill patients, as the induced disease may be difficult to control.A lot of these patients may be receiving many different drugs before transfusion, which may antagonize antimalarial drugs.These facts denote the need for alternative methodologies for the selection of blood to be used in transfusion that could offer fast, cheap and safe results.It is easy, cheap, safe and practical to process donors' blood in vitro with low concentrations of chloroquine.The concentrations given to a patient from a single bag of transfused blood are far low compared to the single dose that might be offered to donors or transfused patients.Thus, it is essential to determine the dose of chloroquine, lethal to the malarial parasites, to be added to donors' fresh blood and to detect the hematological and biological changes that may occur by adding chloroquine to blood infected with malarial parasites and is stored at 4º -6º C.

Materials and Methods
This study was conducted in Ahmed Gasim Hospital, Khartoum State, Sudan.Using a 5ml-chloroquine injection which contains 200mg chloroquine base, three serial concentrations of chloroquine phosphate were prepared as follows: 0.5ml (20mg), 1ml (40mg) and 2ml (80mg) of the injection solution were diluted with normal saline to obtain 80ml of each dilution with 250, 500 and 1000 mg/l concentrations consecutively.Next, fifty microliters of each were dispensed into separate 50ml-blood bags to obtain 250, 500 and 1000µg/l concentrations subsequent to the addition of 50 ml of donors' blood.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 study population if they satisfied the following criteria: 1. Presence of malaria parasite in donors' blood films.2. Density of infection must range between 1000 and 80000 parasites/Jl (only asexual stages were considered).3. The parasites grow when cultured in the first day.4. Donors did not receive quinine within the last 7 days, chloroquine within the last 28 days and Fansidar within the last 14 days.One (200ml) pint (CPDA-1) of donors' malaria parasite infected blood was collected and subdivided equally into four prepared small bags (50ml) using a blood bank mixer (Biomixer-323).Three bags were preloaded with the three concentrations in addition to one bag (control) left without drug.All blood specimens were tested for parasite culture, platelets count, total leukocyte count, packed red cell volume, lysis percentage, osmotic fragility, prothrombin time, activated partial thromboplastin time, sodium and potassium serum levels simultaneously on the day of collection.Thereafter, the bags were stored in the blood bank refrigerator (4º-6ºC) and tested after 24 and 48 hours using the same laboratory procedures.The microscopic malarial parasite identification technique was performed as described by Cheesbrough M. 9 .The absolute number of parasites (number / µL) was estimated in the thick blood films by counting the parasites against 200 white blood cells, multiplied by the total leukocyte count and divided by 200.In vitro cultivation of erythrocytic stages of Plasmodium falciparum was intended for the assessment of the sensitivity of fansidar and to confirm the viability or death of the parasites.It was conducted using RPMI-1640 medium (GIBCO TM ), Invitrogen Corporation.The minimum inhibitory concentration (MIC) was determined by computerized probit/log dose response analysis, SPSS.Indication of resistance was determined by formation of schizonts at 8 pmolchloroquine per well or more.For counting platelets and white blood cells, blood samples were diluted 1 in 20 using (1%) ammonium oxalate and 1 in 20 using (2%) glacial acetic acid solutions respectively.They were counted manually by haemocytometry using the improved Neubauer chamber as described by Cheesbrough M. 9 .Partial thromboplastin time is a screening test for the intrinsic clotting system, i.e. factors XII, XI, IX, VIII, X, V, prothrombin and fibrinogen.Prothrombin time is a screening test for the extrinsic clotting system, i.e. factor VII, X, V, prothrombin and fibrinogen.These tests were performed as described by Dacie& Lewis 10 using the reagent of DiaMedcompany.The osmotic fragility test gives an indication of the surface area/volume ratio of erythrocytes.It measures the effect of the applied doses on erythrocytes' membrane.It was performed as described by Dacie& Lewis 10 .Packed red cell volume is the proportion of whole blood occupied by red cells which was measured as described by Dacie& Lewis 10 .Percentage of lysis is a real indicator for survival of red blood cells during the storage period.Five mls of each blood sample, just after collection, was centrifuged for 5 minutes at 12000g.The supernatant was stored sterile at 4° C to be used as blank.Five hundred microliters of the precipitated red cells was diluted in 5 mls of pure distilled water and stored at 4° C to be used as standard.Both solutions were used in the determination of percent lysis for the same sample throughout the storage period.After 24 and 48 hours one milliliter of each blood sample was also centrifuged for 5 minutes at 12000g and the supernatant of each was read at 540 nm wavelength.Lysis percentage was calculated by dividing the optical density of the tested supernatant by the optical density of the standard and multiplied by 100.
Serum potassium and serum sodium were measured by the flame photometry using Sherwood Scientific-410 flame photometer.Data were analyzed by computer using SPSS program.Non-parametric tests were mainly used for the abnormal distribution of the data.

Results
The total number of individuals who donated blood was 4956; however, 472 (9.5%) of them were rejected on the following bases: 303 (6.1%) were found to be hepatitis B virus carriers while 169 (3.4%) were found to be HIV positive.The collected blood bags totaled 4484.All were subjected to screening for malaria parasites microscopically using a standard Giemsa' staining technique.A total number of 278 (6.2 %) blood bags' specimens were found to carry malaria parasites.The parasite densities were less than a thousand in 215 (77.3%) while parasite-densities of greater than a thousand were observed in 63 (22.7%) specimens.Of these, 30 (10.8%) samples were found to be resistant to chloroquine.In addition, another three (9.09%)samples showed no growth when cultured on the first day.Thus, the actual number of blood samples for this study was reduced according to the above-mentioned parameters to only 30.All of the accepted blood bags belonged to asymptomatic male donors between 25 and 35 years old.
Table (1) demonstrates the reduction of numbers of malaria parasites when different concentrations of chloroquine were applied to donors' blood stored for forty-eight hours.The control of donors' blood samples (without addition of antimalarials) reveals a stable number of parasites even after 48 hours storage.Parasite counts decreased with the increasing concentration of chloroquine and elongation of storage period.Thus the parasites disappear after 48 hours in samples with the higher concentration (1000µg/l) of chloroquine.illustrates the lethal doses of chloroquine.It was observed that higher doses were required when the blood was stored for 24 hours versus 48 hours.Also higher doses of chloroquine were necessary to kill larger numbers of malaria parasites.3 shows the increase in the mean and standard deviation of prothrombin time and partial thromboplastin time with the increase of chloroquine dose and storage duration.Minimum increase was observed when the minimum concentration of the drug was added.
Although there is significant statistical difference between the result times among different blood samples, still the upper levels are within the normal range.4 demonstrates the mean and standard deviation of the osmotic fragilities.It was increased in samples with high doses of chloroquine and with increased storage time.No significant statistical difference was observed between the results of the control and the different blood samples when stored for 24 hours.When blood samples stored for 48 hours, correlation between processed blood samples and the control reveals statistical similarity, still the upper levels are within the normal.Key: C1= 250 µg/l chloroquine C2= 500 µg/l chloroquine C3= 1000 µg/l chloroquineCtro= control Table 5 shows that the packed red cell volume was decreased when the time of storage was prolonged regardless of the increase of the doses.No significant statistical difference was observed between the results of the control and blood samples containing chloroquine during the storage period.6 illustrates the mean and standards deviation of the leukocyte and platelet counts.Each parameter was decreased when the storage time was prolonged and did not correlate with the dose concentration.No significant statistical difference was observed between the results of the control and all of the processed blood samples when they were stored for 24 or 48 hours.7 shows the lysis percentage had increased when the dose of the drug was high as well as when the time of storage was prolonged.No significant statistical difference was observed between the result of the control and blood samples processed with chloroquine.Lysis occurred in blood samples containing each of the antimalarial drugs used in the study is inconsiderable.8 shows no evident increase in serum sodium levelwhen blood samples contain the higher dose but an increase was seen with the increase in the time of storage.
No statistical difference was detected between the results of the control and blood samples processed with 250µg/l chloroquine after 24 and 48 hours of storage nor between samples containing 500µg/l and 1000µg/l chloroquine and the control after 48 hours of storage.Serum potassium levelswere increased when the dose of chloroquine was high as well as when the time of storage was prolonged.After 48 hours no association was found between the results of the control and all blood samples.However, the upper levels of both serum potassium and sodium are within the normal range of the stored blood.
processed blood stored at 4°C is within the normal range.Storage of donors' blood in a blood bank refrigerator resulted in the increase of both sodium and potassium levels in all blood samples including the control, which is likely to be due to the halting of the active transport of these electrolytes across the red cell membrane at 2-8ºC.However, this is reversible after blood is transfused 15 .The lethal doses of the applied drug used in this study appear to be safe for all constituents of the stored blood.Chloroquine was effective and the lethal dose (626.1µg/l)can kill 99% of the parasite within 24 hours.Its application to stored blood did not produce harmful effects on the various components of the blood.Moreover, the doses did not react with the constituents of the blood bag solution (anticoagulant and preservatives) when added before blood collection.Also this procedure is inexpensive; one ampoule of chloroquine is adequate for 800 blood bags.Therefore, in vitro processing of donors' blood with chloroquine appears to be economical and effective.

Conclusion:
In vitro processing of donors' blood with chloroquine is a safe procedure for eradication of transfusion-induced malaria.Chloroquine demonstrated suitable sensitivity against malaria parasites and insignificant effect on the components of the stored blood compared to the infected-control samples without chloroquine.Future studies are recommended to select the most effective and safe antimalarial drug according to the pattern of resistance and side effects.

Table 1
Positivity and mean of the parasite counts during 48-hour storage at 2-8° C

Table 3
Prothrombin time and partial thromboplastin time of the stored blood samples

Table 4
Osmotic fragility values of blood samples during the storage period

Table 6
White blood cell and platelet counts by storage period

Table 7
The lysis percentage of the stored red blood cells during storage at 2-8°C

Table 8
Serum sodium and potassium levels in the stored blood samples