Leishmaniasis in AIDS patients: results of leukocytoconcentration, a fast biological method of diagnosis.

Leishmaniasis in AIDS patients: results of leukocytoconcentration, a fast biological method of diagnosis M.A. Izri,1 M. Deniau,2 C. Briere,1 D. Rivollet,2 J.-C. Petithory,3 R. Houin,2 & J.-J. Rousset1 Leukocytoconcentration is an easy, fast and inexpensive technique for the diagnosis of leishmaniasis from peripheral blood. The technique involves concentration ofblood parasites on a small surface ofa microscope slide while the red blood cells are removed by lysis. The results, compared with those of other methods (examination of cultures of blood samples and bone marrow smears), were very good and accurate. All but one of our cases of leishmaniasis were patients with HIV co-infection. Leukocytoconcentration facilitates follow-up of cases and fast detection of any relapse. Ideally the biological diagnosis of an infectious dis- ease is based on isolation and identification of the pathogenic agent in the host tissue and establish- ment of cultures after direct examination under a microscope. This procedure allows both accurate di- agnosis and an epidemiological survey of the disease. However, when leishmaniasis occurs in an AIDS patient or any other immunocompromised patient, this procedure is often unsatisfactory for several reasons: the samples are difficult to collect, there may be few parasites, and their growth is slow or impeded by other pathogenic agents. The clinical features, when they are not specific, can be attrib- uted to an etiology other than leishmaniasis, and this does not encourage the repetition of laboratory tests (1,2). This report describes an easy, fast and inexpen- sive technique for the diagnosis of leishmaniasis from peripheral blood, and compares the results with those of other techniques. Materials and methods The study, which was carried out during the two years 1993-94, involved 66 patients who were living in Paris or its suburbs but had been infected by leishmaniasis in the Mediterranean area. Most of Laboratory of Parasitology, H6pital Avicenne, 125 route de Stalingrad, 93009 Bobigny, France. Requests for reprints should be sent to Dr lzri at this address. 2 Laboratory of Parasitology, H6pital Henri Mondor, 94010 Creteil, France. 3 Laboratory of Microbiology, H6pital de Gonesse, 95500 Gonesse, France. Reprint No. 5677 them were co-infected with the human immuno- deficiency virus (HIV). Direct blood sample examinations were carried out by the Parasitology Laboratory of the Avicenne Hospital, using a new technique called leukocyto- concentration (LCC) in which the blood parasites are concentrated on a very small surface of a microscope slide, the red blood cells and platelets being removed by lysis. This technique is derived from a single leukoconcentration (3) and one previously used for malaria parasite detection (4), with modifications as described below. The LCC technique (1) Collect a 5 ml blood sample in citrate. (2) Take a blood volume correlated to the number of leukocytes, according to the following formula: volume (in ml) = 1250/leukocytes per ml. (3) Fill a 10 or 15 ml centrifuge tube with this vol- ume and complete with nine volumes of haemolysis solution (0.4% saponin (UCB, Brussels, Belgium), 2.5% formalin, and 0.5% glycerol, made up to 100 ml with physiological saline, filtered twice and stored at room temperature). Using a pipette, aspirate and expell the solution 3 or 4 times over 15 minutes (to mix it properly) before centrifugation. (4) Centrifuge at 840g for 10 minutes (centrifuge: Jouan, Saint-Herblain, France), and pour off the supernatant fluid carefully. (5) Add 100tl of 0.9% saline and dissociate the pellet by homogenization. (6) Cytocentrifuge at 450g for 10 minutes (cyto- centrifuge: Miles Laboratory Inc., IN, USA), the Bulletin of the World Health Organization, 1996, 74 (1): 91-93 K World Health Organization 1996 91 M.A. lzri et al. leukocyte nuclei and leishmanias spread over the limited surface of the slide. (7) Dry the slides and stain with May-Grunwald Giemsa. (8) Examine under a microscope with a x 100 objec- tive after coating the spot with oil. Parasites from the same blood samples were systematically isolated by the Parasitology Labora- tory of the Henri Mondor Hospital using the stand- ard culture technique in Novy, McNeal, Nicolle (NNN) medium overlaid with Schneider's medium and fetal calf serum. Isolation allowed identification of the stocks by isoenzyme analysis. The usual bone marrow, skin and mucosal biopsies were also ex- plored for these parasites, when the clinicians could obtain the necessary samples. Results and discussion A total of 116 leukocytoconcentrations, 12 bone marrow punctures and 128 cultures on NNN medium were analysed from the 66 patients in the study, of whom 12 were cases of leishmaniasis. Blood LCC versus bone marrow puncture (Table 1). Only 12 bone marrow smears were available, be- cause puncture is a traumatic procedure and could not be requested in cases after the diagnosis had already been established. No statistical significance can therefore be deduced from these smears. Never- theless, four positive smears correlated with a posi- tive LCC; in two cases, bone marrow punctures did not allow a diagnosis whereas LCC showed the pres- ence of leishmanias in the blood. Bone marrow punctures cannot easily be repeated and need spe- cialized personnel, which could delay the diagnosis and a medical decision. In contrast, venous blood puncture by a nurse is easy and well tolerated. Parasitaemia occurs with Leishmania donovani (1) and is unusual with L. infantum; but recently in AIDS patients parasites have been found in blood (5, 6), and the LCC technique enhances the possibil- ity of immediate direct diagnosis. Table 1: Results of diagnosis by leukocytoconcen- tration (LCC) and bone marrow smears (BMS) Positive (+) or negative (-) n LCC+, BMS+ 4 LCC+, BMS- 2 LCC-, BMS+ 0 LCC-, BMS- 6 Table 2: Results of diagnosis of leishmaniasis by leukocytoconcentration (LCC) and cultures of blood specimens (C) Positive (+) or No. in No. in negative (-) 1st sample follow-up Total LCC+, C+ 10 23 33 LCC+, C- 2 3 5 LCC-, C+ 0 12 12 LCC-, C- 54 12 66 Total 66 50 116 Blood LCC versus blood cultures (Table 2). The cul- tures are used as control tests. In the present series, there was a strong correlation between the results of LCC and cultures from the same blood samples (P < 0.001), and despite the limited number of samples, the possibility of immediate diagnosis thus obtained is essential for clinicians. Our results must of course be completed by the isolation of the parasites, be- cause their identification is of great interest from both epidemiological and medical viewpoints. Well supplemented NNN medium allows the isolation for identification of the parasite, but the growth of para- sites from blood is slightly slower than that of para- sites isolated from bone marrow punctures. The LCC method is simple and inexpensive and gives very good results, whereas the method of Medrano et al. (7) only permitted the detection of half the positive peripheral blood smears in patients co-infected by leishmaniasis and AIDS. Sophisti- cated techniques such as the polymerase chain reac- tion or antigen detection have been studied and their results seem to be in good agreement with those of other techniques. However, they are expensive and cannot be performed routinely by all laboratories. Besides, they do not allow the isolation of parasites, which is important for sensitivity tests and species identification. The presence and, above all, the abundance of parasites in the blood are certainly a help for diagno- sis, confirming the position of humans in the epide- miological scheme. Formerly, humans were not considered a reservoir host of L. infantum. However, in endemic areas now, an infected subject, especially when co-infected with AIDS, could be a reservoir for sandflies, as suggested by Molina, who infected sandflies using the blood of HIV patients (8). The position of humans in the Leishmania life-cycle is sometimes important; thus, in some foci of L. donovani, humans are an excellent reservoir of infection (9). WHO Bulletin OMS. Vol 74 199692 Leishmaniasis in AIDS patients Conclusion The unreliability of the usual techniques for the diagnosis of visceral leishmaniasis, particularly in immunocompromised patients, led to the search for new methods such as the polymerase chain reaction or circulatory antigen detection. While these are effective, they are expensive and cannot yet be used routinely. Leukocytoconcentration, involving isolation of parasites from the blood, is a process which is both reliable and well tolerated by patients who do not always comply with the need for follow-up examina- tions after therapy. It is capable of making an impor- tant contribution to diagnosis; the financial aspect must also be considered and we suggest a multi- centre evaluation to determine the importance of this technique in the search for parasites from pe- ripheral blood. Resume La leucocytoconcentration, une methode biologique rapide de diagnostic de la leishmaniose chez les malades du Sida. Les auteurs decrivent la leucocytoconcentration (LCC), une technique rapide, sensible et de r6alisation facile pour le diagnostic de certitude de la leishmaniose visc6rale. La LCC est r6alis6e a partir du sang p6riph6rique des patients. Elle concentre les parasites sur la plus petite surface possible d'une lame porte-objet. Ses resultats sont contr6l6s par ceux de la culture sur milieu NNN et compar6s a ceux obtenus par ponction de moelle osseuse. Tous les malades diagnostiques, sauf un, sont coinfectes par le VIH. References 1. Gambarelli F et al. La leishmaniose visc6rale a l'ere du SIDA: les difficultes du diagnostic biologique. M6decine et maladies infectieuses, 1994, 24 (Special): 572-575. 2. Marty P et al. Aspects biocliniques de la leishmaniose visc6rale chez les sideens. Bulletin de la Societ6 de Pathologie exotique, 1989, 7: 159-161. 3. Izri MA et al. La leishmaniose visc6rale: diagnostic par leucoconcentration. La presse m6dicale, 1993, 22 (No. 21): 1010. 4. Petithory JC et al. Microscopic diagnosis of malaria following a cytoconcentration technique after saponin treatment to cause hemolysis. American journal of tropical medicine and hygiene, 1993, 49: 245 (abstract). 5. Fillola G et al. Peripheral intramonocytic leishmanias in AIDS patients. Journal of clinical microbiology, 1992, 30: 3284-3285. 6. Martinez P et al. Diagnosis of visceral leishmaniasis in HIV-infected individuals using peripheral blood smears. AIDS, 1993, 7: 227-230. 7. Medrano FJ et al. An easy and quick method for the diagnosis of visceral leishmaniasis in HIV-1-infected individuals. AIDS, 1993, 7: 1939. 8. Molina R et al. Isolation of Leishmania infantum from the blood of a patient with AIDS using sandflies. Trans- actions of the Royal Society of Tropical Medicine and Hygiene, 1992, 86: 516. 9. Chulay JD, Adoyo MA, Githure JD. Leishmania donovani parasitaemia in Kenyan visceral leishmaniasis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 1985, 79: 218-222. WHO Bulletin OMS. Vol 74 1996 93