QBC ® for the diagnosis of human and canine american visceral leishmaniasis : preliminary data QBC ® para o diagnóstico de leishmaniose visceral americana humana e canina : dados preliminares

Quantitative Buffy Coat” (QBC®) is a direct and fast fluorescent method used for the identification of blood parasites. Since Leishmania chagasi circulates in blood, we decided to test it in American visceral leishmaniasis (AVL). Bone marrow (BM) and peripheral blood (PB) of 49 persons and PB of 31 dogs were analyzed. QBC® was positive in BM of 11/11 patients with AVL and in 1/6 patients with other diseases. Amastigotes were identified in PB of 18/22 patients with AVL and in none without AVL. The test was positive in 30 out of the 31 seropositive dogs and in 28/28 dogs with Leishmania identified in other tissues. QBC® is a promising method for diagnosis of human AVL, and possibly for the exam of PB of patients with AVL/AIDS, for the control of the cure and for the identification of asymptomatic carriers. Because it is fast and easy to collect and execute, QBC® should be evaluated for programs of reservoir control. Key-words: Kala-azar. Visceral leishmaniasis. Leishmania chagasi. Dog. QBC®. 1. Laboratório de Leishmanioses, Hospital de Doenças Infecto-Contagiosas, Departamento de Medicina Comunitária, Universidade Federal do Piauí. 2 . Laboratório de Sanidade Animal, Departamento de Clínica e Cirurgia Veterinária, Universidade Federal do Piauí. 3 . Coordenação Geral de Laboratórios de Saúde Pública, Fundação Nacional de Saúde, Ministério da Saúde. Logistic and financial support: Coordenação Geral de Laboratórios de Saúde Pública e Coordenação Regional no Piauí, Fundação Nacional de Saúde, Ministério da Saúde and Programa Institucional de Bolsas de Iniciação Científica, CNPq (DBL). Address to: Dr. Carlos Henrique Nery Costa. Laboratório de Leishmanioses/Hospital de Doenças Infecto-Contagiosas. Rua Artur de Vasconcelos 151-Sul, 64.000-450 Teresina, PI, Brazil. e-mail: crlshncst@aol.com Recebido para publicação em 5/7/2001. The diagnosis of human and canine American visceral leishmaniasis (AVL) may be confirmed by serologic tests or by the demonstration of the protozoa Leishmania chagasi with parasitological or molecular methods. Both approaches present drawbacks that may be improved. While false-positive results may appear with serology due to cross-reactivity or due to the development of specific antibodies to past asymptomatic infections, parasitological methods are invasive because they usually require bone marrow (BM) or spleen puncture, and polymerase chain reaction (PCR) from peripheral blood still lacks feasibility and reliability for routine diagnosis17. Stimulated by the recent description of high proportion of Leishmania DNA in the peripheral blood (PB) of patients with AVL25 and by the need of a diagnostic method for canine AVL which directly identifies the parasites, we have proposed the use of the Quantitative Buffy Coat (QBC®). The method, which is largely used for diagnosis of malaria15 32, was intended for rapid demonstration of amastigotes in the BM and PB of persons with AVL and in dogs infected with L. chagasi. In this paper we report the preliminary but larger data of our previous observations of QBC® for human and canine AVL in Teresina, Brazil20.

The diagnosis of human and canine American visceral leishmaniasis (AVL) may be confirmed by serologic tests or by the demonstration of the protozoa Leishmania chagasi with parasitological or molecular methods.Both approaches present drawbacks that may be improved.While false-positive results may appear with serology due to cross-reactivity or due to the development of specific antibodies to past asymptomatic infections, parasitological methods are invasive because they usually require bone marrow (BM) or spleen puncture, and polymerase chain reaction (PCR) from peripheral blood still lacks feasibility and reliability for routine diagnosis 17 .
Stimulated by the recent description of high proportion of Leishmania DNA in the peripheral blood (PB) of patients with AVL 25 and by the need of a diagnostic method for canine AVL which directly identifies the parasites 10 , we have proposed the use of the Quantitative Buffy Coat (QBC®).The method, which is largely used for diagnosis of malaria 15 32 , was intended for rapid demonstration of amastigotes in the BM and PB of persons with AVL and in dogs infected with L. chagasi.In this paper we report the preliminary but larger data of our previous observations of QBC® for human and canine AVL in Teresina, Brazil 20 .

SUBJECTS AND METHODS
BM and PB of persons and dogs were examined using QBC®.Persons' samples were taken from hospitalized patients with AVL, from hospitalized patients with other diseases and from healthy volunteers.The diagnosis of AVL was established when typical symptoms and reactive immunofluorescent antibody test (IFAT) (Biomanguinhos-RJ) or amastigotes in the BM aspirate were present.Seropositive dogs screened with IFAT (dilutions 1/40 or more) were examined by a veterinarian (ILM).Because they should be killed according to the recommendations of the Ministry of Health, they could be autopsied.PB was examined using QBC® and samples of BM, spleen, liver, lymph nodes and kidneys were stained with Giemsa and cultivated in the Novy-MacNeal-Nicolle (NNN) media.
BM and PB were placed on a 75-mm (55ml) microhematocrit capillary tube previously coated with DNA stain acridine orange and potassium oxalate.Centrifugation at a preset speed (14,000 x g) (Parafuge, Becton Dickinson) allowed the separation of blood cells, with the help of a polystirene float for expansion of the white cell layer.To adapt a benchtop microscope into a fluorescent microscope, the oil immersion objective (Paralens UV Microscope Adapter, Becton Dickinson) was screwed to a portable light source in the range of 480nm by a fiberoptic cable.Microphotographs were taken using a Zeiss® microscope with epiimmunofluorescence HBO-50 and with a microphotographic system MC 80 DX and 1,000x amplification.A fluorescent microscopic was also ocasionally used, although a little more cumbersome to get the focus.
The first part of the study consisted in practice and training.A skilled technician on malaria diagnosis with QBC® (FCOL) examined human and dog samples while teaching the Teresina team.At the same time, the pattern of fluorescence of the L. chagasi nuclei and kinetoplast on QBC® of promastigotes on NNN culture tubes was observed and characterized.When these flagellated forms found in sand flies and in culture change into the non-flagellated amastigotes found on host tissues, there is no morphological modification of the nuclei or the kinetoplast.Therefore, both forms are undistinguishable at QBC. Due to the initially high falsepositive rate, a set of guidelines for amastigote identification was used in order to increase stringency; to be taken as positive, a sample must show all of the following 5 characteristics: parasite nuclei looking less then 1/5 the size of nuclei of host cells (1), not presenting cytoplasmic fluorescence (2) and showing the presence of the kinetoplast, which must border the parasite nuclei (3), being brighter than it (4) and seeming not to be more than 1/3 of its size (5).RESULTS The typical aspects of PB extracellular amastigotes and BM intracellular forms are seen in figure 1. Parasites were identified in the less dense cell layer.While PB preparations were clear and neat, BM tubes yield longer cell layers and were full of nucleated cells.Only one intracellular parasite was clearly seen on PB, but collections of parasites suggestive of being extracellular, were identified.
Before the establishment of the guidelines for parasite identification, initial PB examination of patients with AVL detected amastigotes in 16/19 (84%).Only 16/26 (62%) of the BM tested were positive.Four patients thought not to have AVL presented false-positive results on PB.Table 1 presents the results of studies on 49 persons after the parameters for taking the samples as positive were used.All patients with AVL had positive QBC® on BM examination.One patient with splenomegaly had positive QBC® on BM but histopathology lately defined the diagnosis as paracoccidioidomycosis.
All 28 dogs in which parasites were found presented positive QBC® on PB.One dog with suggestive symptoms but without parasites identified on authopsy had a negative result.The overall sensitivity of the test was 97% (Table 2).Because no trial was designed for it, specificity of QBC® on PB for canine AVL was not evaluated in this study.DISCUSSION A simple fluorescent body on QBC® may not represent the nuclei of extracellular amastigotes, as the initially high false positive rate has shown.In order to increase the specificity, typical morphological characteristics were required, which, in fact, improved the test.When the pattern of fluorescence, the size of the nuclei and the presence of the kinetoplast were considered, the proportion of false-positive tests was significantly reduced.Only one patient tested falsepositive.This patient presented fever, anemia and splenomegaly with the disseminated form of the South American deep mycosis caused by Paracoccidioides brasiliensis.The fungi was mistaken for Leishmania on BM QBC®, but this is not surprising since the shape of a fluorescent yeast forming the characteristic bud might mimic the shape of an amastigote.However, although caution should be taken on the interpretation of a BM positive QBC®, the high sensitivity on BM and the high pre-test probability of AVL in patients with typical symptoms would balance the moderate specificity and allow the indication of BM QBC® for routine use.
QBC® had a satisfactory performance on human blood examination.Indeed, although these data are not new, in recent years Leishmania has been repeatedly demonstrated, either directly, culturing or via PCR®, in PB from asymptomatic persons infected with anthroponotic and zoonotic viscerotropic Leishmania 13 27 and from immunocompromised 11 22 and immunocompetent 1 28 30 31 Old

Figure 1 -
Figure 1 -Microphotographs of QBC® and Giemsa stain from bone marrow and peripheral blood of humans and dogs taken in a microscope of epifluorescence.Top left: Human bone marrow.A single amastigote is visible among many nucleated cells; the kinetoplast is situated at the position of 12:00 o'clock in relation to the parasite nuclei.Top right: Human peripheral blood with one isolated amastigote; the kinetoplast is found at the position of 5:00 o'clock.Middle left: Human peripheral blood with a group of extracellular amastigotes; the kinetoplast is visible in some of them.Middle right: Cluster of intracellular amastigotes from human peripheral blood.Bottom left: Dog peripheral blood with amastigotes; the kinetoplast is discernible in some parasite cells.Bottom right: Giemsa stain of dog bone marrow with several amastigotes, to highlight the similarity and differences of both staining methods.Magnification 1,000x.

Table 1 -
QBC® results from bone marrow and peripheral blood in persons with and without American visceral leishmaniasis.

Table 2 -
QBC® results in seropositive dogs accordingly to the identification of Leishmania chagasi at authopsy and to the clinical status.