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On-line version ISSN 1806-4841
An. Bras. Dermatol. vol.77 no.5 Rio de Janeiro Sept./Oct. 2002
CLINICAL, LABORATORY AND THERAPEUTIC INVESTIGATION
Comparative study of parasitological techniques for demonstration of amastigotes and primary isolation of promastigotes in American Cutaneous leishmaniasis*
Raimunda Nonata Ribeiro SampaioI; Gilberto Brown de Andrade II; Antonio César PereiraIII; Eurico Aparecido da SilvaVI; César Augusto Cuba CubaV
Professor of Dermatology, Coordinator of the Dermatology Laboratory, Head of
the Dermatology Service of the Hospital Universitário de Brasília
(HUB)-Universidade de Brasília (UnB)
IIResident M.D. of the Hospital Distrital de Brasília Medical Clinic, and Alma Mater of UnB
IIIM.D., Alma Mater of UnB
VIM.D., Alma Mater of UnB
VTitular Professor of Parasitology, Coordinator of the UnB Parasitology Laboratory
PCR is a highly sensitive procedure for the diagnosis of LTA, but it is expensive
and not readily available. Culture and smearing are practical, but they lack
OBJECTIVE: The objective of this work is to compare the latter two methods in order to achieve a higher sensitivity at a lower cost.
METHOD: Three culture media were compared in the process of isolating leishmaniasis: blood Agar Difco + Schneider + 20% fetal calf serum; Difco + Schneider + human sterile urine (2%); Schneider with sterile human urine (2%). Moreover, two techniques were compared for the demonstration of amastigotes: biopsy performed with smears, or matchstick scrapping
RESULTS: The positivity and contamination indices (29-to-33% and 8-to-11%, respectively, p>0.05) were similar when comparing the cultures. The smears carried out by biopsy or the matchstick scrapping procedure had no significant differences (14 and 19%, respectively, p>0.05). Leishmania (Viannia) braziliensis predominated.
CONCLUSION: In Brazil, urine may substitute fetal calf serum. The cost-to-benefit ratio is advantageous. Urine has no cost, while the market value of 500 ml of fetal calf serum is $US 185.
Key words: Diagnosis; Leishmania braziliensis; Leishmaniasis, cutaneous.
The laboratory diagnosis of American Cutaneous Leishmaniasis (ACT) may be performed by the aid of parasitological tests, immunologic proofs and molecular biology methods. In parasitological tests, evidence of parasites was sought by means of direct and indirect tests for the purpose of confirming the cause of the disease.
Two important parasitological methods are laminated smears, using the tissue fragment obtained by biopsy or by matchstick scrapping of the lesion,1and growth of Leishmania in culture obtained by the inoculation of material withdrawn from the lesion aspired by punch biopsy or by triturated biopsy. Both methods have a lower percentage of parasite findings, mainly regarding Leishmania (Viannia) braziliensis.2,3,4 Smear tests for revealing the parasite are important because they are easily performed and may provide rapid diagnosis if the slide preparation is done correctly. The finding of parasites is more successful in culture sampling than by smear. Parasitiological research by inoculation in hamsters, in spite of being more sensitive, is slow and onerous. Performing three aspirations at different lesion sites increases the sensitivity of isolating promastigotes in cultures of the cutaneous lesions.2,3
Howard et al. showed that human urine stimulates cell division in vitro of Leishmania, possibly facilitating the primary culture of infected cells5 Armstrong et al. reported that sterile human urine added to the culture medium also stimulates cell division in promastigote forms and facilitates the primary culture of infected animal tissue cells, possibly substituting calf fetal serum in culture media.6 Previously identified strains maintained in cryopreservation were used in both cases.
The objective of this study was to verify whether sterile human urine added to the Difco and Scheneider medium would be more efficient or could substitute fetal calf serum in Leishmanias species cultures available in the country. The second objective was to determine whether the smear techniques, in association with material collected by biopsy or matchstick scrapping technique, could have different sensibilities.
PATIENTS AND METHODS
A selection was made of 21 patients with ACT cutaneous lesions undergoing care at the outpatient clinic specialized in the disease located at the Hospital Universitário de Brasília (HUB), Universidade de Brasília. All patients were submitted to Montenegro's reaction, indirect survey of fluorescent antibodies, histopathological test and later skin lesion treatment. They had been previously informed of the work, to which they gave their consent. Also included were patients with skin lesions compatible with ACT and who had, apart from positive Montenegro's intradermoreaction, at least another positive specific test: smear, culture, IFI or histopathologic test. Asepsis of lesions was performed with 0.9% physiological serum, iodized alcohol and 95o alcohol, in this sequence. The aspired sites and biopsies were anaesthetized with 2% lidocaine containing adrenaline, excluding the latter when treating the extremities. Using a syringe mounted with a 25 x 8 mm needle, containing 0.5 ml of saline solution with gentamicin (100 mcg/ml), introduced under light pressure on the edge of the integral and infiltrated lesion surface, the material was aspirated and inoculated in the culture tubes.
Three different compositions were used in the culture media: 1. Blood Agar Difco + with 0.5 ml of Schneider (20% fetal calf serum) with (D+S+SB) supernatant; 2. similar medium to the former increased with a 2% sterile human urine (U) concentration filtered with a bacterial millipore using a 0.45m filter (D+S+SB+U); 3. a medium containing Schneider (with fetal calf serum) supplemented with 5% U (S+U). All media were dosed with gentamicin in a 100 mcg/ml concentration. Four aspirations were performed on each lesion, which were inoculated randomly in three tubes containing the D+S+SB medium, three with D+S+SB+U and two containing S+U. Culture media, after inoculations, were incubated at 23ºC +/-1ºC and examined weekly under an inverted light microscope for 30 days, following which the negative tubes were disconsidered.
In the technique showing amastigotes, the biopsy was performed on the infiltrate edge and over the whole lesion surface, using 4-mm diameter punch. The fragment was compressed between two slides, after withdrawing excess blood with filter paper. The slides were allowed to dry over the ensuing period for two-to-three minutes at room temperature. Afterward they were fixed with methyl alcohol (methanol) for four minutes and colored by Giemsa and, later, examined by means of immersion oil under the optical microscope (x 100) for 15 minutes.
At the internal margin of the same lesion, scarring was made by using a wooden scrapper stick with a previously autoclavated matchstick edge. The material collected was spread over two slides per patient, submitted to the same aforementioned sequence for collecting material for these cultures. The statistical method applied was the Chi-squared test, using the 2 x 2 type table analysis for verifying group differences.
Of the cases studied 77% had zero-to-four month progression, 19% five-to-eight months, and 4% had more than eight months of disease progression.
The techniques for isolating the promastigotes in three media showed positivity indices and similar contamination (29-to-33%, and 8-to-11%, respectively). (Table 1). The techniques showing the amastigotes between the smear performed by punch biopsy and the matchstick method also had similar positivity indices (14 and 19%, respectively). When compared with the Chi-squared test, the results of the various culture media and the amastigote research showed no significant difference.
DISCUSSION AND CONCLUSION
This diagnosis confirms verification of parasite presence in the lesion suspected of having ACT. With the techniques available in practical use, however, successful findings of Leishmania are represented by a low percentage (18-to-52% for smear research).1,7 Finding parasites became even more difficult in cases where the etiological agent is Leishmania (Viannia) braziliensis, as well as when the lesion would be mucosal.4 It was almost impossible to find it in the chronic verrucosa and mucosal cutaneous forms, demonstrating little disease activity.
Nowadays, there are highly sensitive methods in molecular biology, like PCR, for the diagnosis of ACT. However, this technique requires specialized equipment and adequate infrastructure. Its execution is of little practical use as well as being onerous, which is why it is still a long way from daily practice. Yet today the authors believe it is important to find ways within the scope of a country's possibilities to improve the methods available at all levels of health care which promote better therapy orientation and improved disease prognosis.
At the HUB, where these cases were studied, patients came predominantly from the states of Goias, Minas Gerias, Bahia and the Distrito Federal. Most of the reference hospital cases are L (V) braziliensis, followed by L (L) amazonensis, with other species rarely seen, such as L(V) shawi.9,10 This constitutes a good model by which to test the species that predominate. The preponderance of L (V) braziliensis justifies low indices in finding Leishmania in the results. As for contamination indices, they were acceptable for the culture of all media employed.
As mentioned, the authors had already confirmed the effectiveness of human urine for amastigote growth in cell cultures and for promastigotes maintained in the laboratory, whose origin was either oriental countries or unknown. In the research reported in this paper the cultures were performed on material collected from the patient and evaluated in terms of species that predominate or exist in the country, such as (L(V) braziliensis, L(L) amazonensis and L(V) shawi). This led the authors to conclude that human urine may substitute fetal calf serum in Brazil. This is an advantageous alternative when its cost-benefit ratio is considered. A 500-ml flask of fetal calf serum costs $185 on average, whereas human urine has no cost whatsoever.
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6. Armstrong TC, Patterson JL. Cultivation of Leishmania braziliensis in an economical Serum-free Medium containing human urine. J Parasitol 1994; 80: 1030-2. [ Links ]
7. Navin TR, Arana FE, Merida AM, Arana BA, Castilho AL, Silvers N. Cutaneous leishmaniasis in Guatemala: Comparison of diagnostic methods. Am J Trop Med Hyg 1990; 42: 36-42. [ Links ]
8. Laskay T, Mikó TL, Negesse Y, Solbach W, Rollinghoff M, Frommel D. Detection of cutaneous Leishmania infection in paraffin-embedded skin biopsies using the polymerase chain reaction. Trans R Soc Trop Med Hyg 1995; 89: 273-5. [ Links ]
9. Paula, CDR. Estudo comparativo entre isotionato de pentamidina, administrada em três doses, com o esquema usual de metil meglumina para o tratamento da forma cutânea de leishmaniose tegumentar americana. Tese. Brasília: Universidade de Brasília, 1999. [ Links ]
10. Sampaio, RNR, Marsden PD, Furtado T, Barreto AC, Cuba CC, Campbell, GMA.Avaliação clínica e laboratorial de 114 casos hospitalares de Leishmaniose cutâneo mucosa. An Bras Dermatol 1989; 64: 201-5. [ Links ]
Profa. Raimunda Nonata Ribeiro Sampaio
SHIS QI 25, Conj 2, casa 1, Lago Sul
Brasília DF 71660 220
Tel.: (61) 367-1331
Fax: (61) 367-3825
Received in July,
5th of 2001.
Approved by the Consultive Council and accepted for publication in April, 16th of 2002.
*Work done at "Dermatology Service of the HUB; UnB Dermatology laboratory and Parasitology laboratory".