Continuous-flow apheresis of microfilariae in Loa loa infestations

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<ul><li><p>Plasma Tber Transfus Technol 1988; 9:95-98 0278~6222/88 $3.00+0.00 Printed in Great Britain Pergamon Press plc </p><p>Continuous-Flow Apheresis of Microfilariae in Loa Loa </p><p>Infestations Peter Kern </p><p>Peter Wind Manfred Dietrich </p><p>m Loiasis is endemic in the tropical rain forest of Central and some parts of West Africa and is transmitted by the insect vector Chrysops silacea and Chrysops dimidiata. Loa loa infestation causes lo- cal inflammation due to migrating adult worms in the subcutis (Calabar swelling) and in the conjunctivae (eye worm). The diagnosis is made by the demonstration of circulating blood microfilariae, the lar- vae of the adult worms. Since the density of the microfilariae is very low, they can only be identified by concentration methods, such as hemofiltration. During treatment with the specific antiparasitic drug (diethylcarbamazine, DEC), side ef- fects such as allergic reactions due to the rapid disruption of circulating microfilar- iae may occur. Those symptoms are aggra- vated in patients with a high number of larvae, leading to meningo-encephalo- myelitis or even death. 1 </p><p>Since microfilariae accumulate in the huffy coat during leukocytapheresis, 2 it was thought that this technique might be a suitable tool to reduce the parasite load and to bypass the side effects of specific drug treatment. Muylle et al. achieved apheresis of microfilariae in two patients with Loa loa infestation by using a Haemonetics, M-300 blood processor. 3 </p><p>From the Climcal Department, Bernhard-Nocht.Instttut fur Schtffs- und Tropenkrankhelten, Bernhard-Nocht-Str 4 ZOO0 Hamburg 4. West Germany </p><p>Re rmt requests to Prw Doz. Dr. I? Kern, Khmsche Ab- tel ung. Bernhard-Nocht-Institut. Bernhard.Nocht-Str 4, P ZOO0 Hamburg 4, West Germany. </p><p>9s </p><p>The authors studied two patients with an excessive microfilarial count in the peripheral blood of up to ~/FL and achieved a high enrichment by discontinuous-flow centrifugation. A similar observation was reported by Saeed et al. later on.4 We have studied two pa- tients with loiasis and report the results of microfilarial apheresis using a con- tinuous-flow IBMlCobe cell separator. W </p><p>PATIENTS AND METHODS </p><p>A 29-year-old German woman, patient 1, undertook an adventure tour in 1980 through all parts of West Africa. After returning, no clinical symptoms of hel- minthic diseases were noted. However, 18 months later headache, urticaria, and recurrent edema of the left eye led to con- sultation with an ophthalmologist who referred the patient to our clinical depart- ment (Figure 1). Diagnosis of loa loa was made by demonstration of high numbers of microfilariae in the peripheral blood (hemofiltration) and thick blood film (Figure 2). Laboratory findings revealed a marked leukocytosis (2O,OOO/~L) and eo- sinophilia (56%), a highly elevated IgE level of more than 1000 ID/r&amp; and anti- bodies against common filarial antigen (Dirofilaria immitis). Bvo aphereses were performed before specific therapy was be- gun in October 1983. Centrifugation was done around midday in order to coincide </p></li><li><p>96 Plasma Ther Transfus Technol Vol. 9, No. 1 </p><p>Figure 1. Migration of Loa loa in the eyelid. </p><p>with the period of maximum numbers of microfilariae in the peripheral circu- lation. </p><p>From October 1980 to September 1983 a 32-year-old German engineer, pa- </p><p>Figure 2. Numerous microfilariae in the buffy coat. </p><p>tient 2, worked in Nigeria for a German construction company. During a routine medical check-up, there were no specific complaints, no itching, and no Calabar swelling, but an increased number of eo- sinophils were detected in the blood film. Therefore, several investigations were per- formed in our department to establish the diagnosis. Loa loa microfilariae were de- tected in the blood by concentration tech- niques. Laboratory results showed a leukocytosis of 13,4OO/pL and eo- sinophilia of 59%. Highly elevated anti- body levels against common filarial antigen (Dirofilaria immitis) and slightly elevated IgE level were found. Four runs of continuous-flow apheresis were per- formed before specific therapy was begun in October 1983. </p><p>An IBM 2997 continuous-flow cell separator was used to treat these patients. The parameters chosen for leukocytaphe- resis were: 600 to 800 rotations per minute, a separation time of two hours, and a processed blood volume of 4 to 8 liters. In all runs, citrate was used as anti- coagulant, in addition to hydroxyethyl starch (molecular weight 250,000) in order to enhance erythrocyte sedimenta- tion. The buffy coat was collected con- </p></li><li><p>Apheresis in Loa Loa Infection 97 _ </p><p>tinuously into tubes. White blood cells and microfilariae were counted in each tube [volume of approximately 40 mL) by standard methods. In addition, enumera- tions were performed before, during, and after the procedure. </p><p>RESULTS AND DISCUSSION </p><p>By continuous-flow centrifugation, Loa loa microfilariae were harvested in the buffy coat layer. Our two patients with Loa loa infestation differed with regard to the initial density of microfilariae in the peripheral blood. Prior to centrifugation, high numbers of larvae were detected in Patient 1 in contrast to low numbers in Patient 2. The efficiency of microfilarial apheresis closely paralleled that of the leukocyte yield during several runs as shown in Figure 3 A and B (patient 1, run B, patient, 2, run B, respectively). At- tempts to optimize the harvest of microfilariae by variation of the speed of centrifugation were not successful. In subsequent leukocytaphereses performed in the two patients, the yield of micro- filariae decreased considerably, as shown in Table 1. The procedure was well toler- ated; however, suboptimal venous access in Patient 1 made further aphereses impossible. </p><p>Continuous apheresis of microfilar- iae was achieved in our two patients with Loa loa infestation. Muylle et al.3 and Saeed et al.4 reported on a similar ap- proach using the discontinuous-flow cen- trifugation technique. Microfilariae were </p><p>efficiently harvested from the buffy coat layer. Some variations of the microfil- ariae yield were noted during continuous- flow centrifugation. A number of varia- bles might explain this finding, such as enumeration techniques of larvae, adhe- sion or clumping of microfilariae in the tubing system, or in vivo shift of the para- site in response to the procedure. Skillful parasitological methods and apheresis technology must be combined to obtain benefit for patients with excessive num- bers of circulating microfilariae. In these instances, apheresis has a place in the management of loiasis, as has also been pointed out by others. 3.4 This technique might also be used to harvest a large num- ber of microfilariae for in vitro studies, such as antigen preparation for serologic investigations. In addition, this method might be applied successfully to those pa- tients with clinical symptoms of filaria- sis but no detectable microfilariae by standard diagnostic concentration or filtration methods. In this case, leu- kocytapheresis can facilitate the species- specific diagnosis as early as possible, since corresponding antibody tests are not available. </p><p>Acknowledgments </p><p>This study WLIS supported by the Bundesministerium fiir lugend, Familie. Frauen und Gesundheit, Bonn, West Germany. </p><p>REFERENCES </p><p>1. Brumpt LC, Pequingnot H, Lhermitte F, Petithory J, Remy H: Loase avec </p><p>nble 1. Leukocytapheresis in Two Patients with Loa Loa Infestation: T%hnical Detaih and Results of Repeated Investigations </p><p>Ptocessed Flow Yield of Yield of Separation RPM Blood Volume (L) mLlmin Leukocytes x lOlo Microfilariae x lo3 Patient 1 </p><p>Run A 800 1.4 25-30 0.9 99 Run B 600 6.0 35-50 3.4 17.5 </p><p>Patient 2 Run A 800 3.75 45 1.4 4.34 Run B 600 6.0 60 2.8 4.08 Run C 650 7.22 70 2.9 0.39 Run D 800 5.46 60 1.59 0.02 </p><p>PLASIAT 9:1-G </p></li><li><p>98 Plasma Ther Transfus Technol Vol. 9, No. 1 </p><p>I; 80 Rpm - 800 2. flow-35-50 ml/min </p><p>3. - ._ =: E200 : .Y Pat 2 run </p><p>01 = 150 Rpm-BOO </p><p>110~ -00 m </p><p>120 </p><p>50 </p><p>40 </p><p>n Lib b </p><p>llmin </p><p>4. </p><p>-1 2 3 4 5 6 itr. </p><p>ptocrr~rd blood volunr </p><p>Figure 3. Leukocyte and microfilariae enumerations during continuous-flow centrifugation. Similar separation conditions are illustrated (panel A patient 1, panel B patient 2). </p><p>microfilaremie Clevee, encephalite ther- apeutique, traitement par exsanguino- transfusion. Bull Mbm Sot Mad Hepit Paris 1966; 117:1049-1058. Buckner D, Eisel R, Perry S: Blood cell separation in the dog by continuous-flow centrifugation. Blood 1968; 31:653-672. Muylle L, Taelman H, Moldenhauer R, Van Brabant R, Peetermans ME: Useful- ness of apheresis to extract microfilarias in the management of loiasis. Br Med \ 1983; 2:519-520. Saeed AA, Green PJ, Naoroz M, Lee HA, Venkat Raman G: Loa loa: The use of a blood cell separator to reduce microfilarae- mia before specific chemotherapy. I Infect 1984; 9:161-166. </p></li></ul>


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