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vol.21 número3-4STUDIES OF THE LEISHMANIN SKIN TEST POSITIVITY IN CASES WITH TREATMENT ANTI-Leishmania índice de autoresíndice de assuntospesquisa de artigos
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Parasitología al día

versão impressa ISSN 0716-0720

Parasitol. día v.21 n.3-4 Santiago jul. 1997

http://dx.doi.org/10.4067/S0716-07201997000300001 

TRABAJOS DE INVESTIGACION

LOCALIZED CUTANEOUS LEISHMANIOSIS
USING POLYMERASE CHAIN REACTION:
A VENEZUELAN FAMILY REPORT

ANA LUGO DE YARBUH, GLORIA PREMOLI-DE-PERCOCO y MANUEL VALERA

ABSTRACT

In this study, we have tested the sensitivity of a specific probe such as the polymerase chain reaction (PCR), as an alternative approach to parasitological methods to evaluate patients with cutaneous leishmaniosis (CL). The assay was designed to evaluate familiar group with an infection of 4 months. The patients were diagnosed according to clinical criteria, parasitological, immunological assay and PCR specific probe. Five of the family group reported a clinical picture with a lymphadenopathy, localized CL lesions with 2, 15, 27 30 and 60 cutaneous lesions and with different size (mean=5x7 for 27x35mm of diameter), detectable on the exposed body surface: face (cheek, nose, forehead, front), neck, shoulder, hand, forearm and foot. The 6 patients were diagnosed by leishmanin skin test (LST) (mean = 26.2x28.4 mm of diameter). Anti-Leishmania specific antibody were detected by serodiagnosis by the direct agglutination test (DAT),immunofluorescence antibody test (IFAT) and Enzyme-linked immunosorbent assay (ELISA) in all patients. The Leishmania parasite were evident in the lesions of 5 patients, using Giemsa-stained of smear specimens from the lesion, imprint of biopsies lhe lesion, culture and inoculation in hamster of lesions material, sections histological by conventional stained, immunostaining techniques and by PCR assay on serum samples. The PCR procedure increased the sensitivity of direct microscopy diagnostic of CL, therefore should be considered as a valuable and sensitive diagnostic tool in the identification of species of Leishmania and in the diagnosis de CL in the asymptomatic patients.

Key words: Polymerase Chain Reaction, Leishmania braziliensis, diagnosis.

 

Acknowledgements. This work was financially supported by C.D.C.H.T.-ULA, Grant C-747-95-03.b. Grant C –642-94-03-A. Grant 040-95-B-07. Grant 04-97-A-07. Grant 04-97-C-07.

 

INTRODUCTION

The leishmaniosis constitutes a group of disease with clinical manifestations in human that may vary from simple cutaneous lesions to mucocutaneous ulcers or visceral diseases. The dermatological forms caused by Leishmania genus in the New World are referred as american cutaneous leishmaniosis (ACL) and it is initiated by inoculation intradermal of Leishmania promastigotes during a blood meal of an infected sand fly. The clinical response to Leishmania in humans depend on parasite and host variation susceptibility, there may be a latent period between the LST conversion and clinical response1. The parasite go into the mononuclear phagocytes of the skin, involvement this organ in the early immune response might be relevant to the outcome of the disease. An adequate cell-mediated immune response is mounted against the parasite, in consequence a complex host immunological response with depending upon the infecting species of protozoan parasites involved, and presentation of the disease is restricted to well defined skin lesion2, 3. The disease is often self-limiting, the lesions healing spontaneously after a period of several months with develop of delayed hypersensitivity, but other patients require specific therapy4-6. These variations in the immune response have made in leishmaniosis and excellent prototype for studying the immunoregulatory processes involved in infectious diseases7.

In this study, we distinguish clinical infections and an asymptomatic patients, by the development of molecular tools, using the PCR probe for detecting L. braziliensis DNA in tire blood of patients with CL.

MATERIALS AND METHODS

Study group

A clinical history of a family group of 6 patients was conducted with suspected localized CL with 4 months of evolution. One of the cases has no clinical evidence of CL. They are from endemic area of Mérida state Venezuela. The LST was carried out on each patient, with a dose of 0.1 ml of promastigotes to a concentration of 105 cell/ml, injected intradermally on the inner surface of the left forearm and read 48 h later.

Serodiagnosis

Anti-Leishmanial antibodies were determined in the serum collected at cases and screened for specific antibody, using a formalin-treated promastigote antigen prepared from L. garnhami strain H/HOM/Ve576JAP. The DAT8, IFAT using an anti-human polyvalent immunoglobulins FITC9 and ELISA with an anti-human immunoglobulins peroxidase conjugate second antibody10 were assay.

 

Identification of Leishmania parasites

Parasitological confirmation of the clinical diagnosis was based in the visualization of parasites by microscopic analysis of Giemsa-stained smears from aspirated material from skin lesion, tissue imprints and histological sections.

Isolation of the parasites

Culture of minced biopsy material was transferred to bifasic culture NNN, incubated to 26ºC and examined for promastigotes.

Biopsy macerate in phosphate buffer saline (PBS) to pH 7.2, and about 0.2 to 0.5 ml of the homogenate was injected intradermally in the feet the hamsters. The animals were killed 10 weeks later and podal infected tissues and the samples of spleen and liver were homogenized in PBS under sterile conditions. Aliquots of the homogenate were promptly transferred to culture NNN, incubated to 26ºC and examined for promastigotes.

Histopatology and lmmunostaining assay

Biopsies were taken and fixed in formalin to 10% for 2 h, and then transferred to 70% ethanol until processed. Seven micras paraffin wax-embedded sections was prepared for the demonstration of Leishmania antigen for the histopatological examination of Giemsa-Colophonium (GC) stained section11, unlabelled peroxidase-antiperoxidase (PAP) and IFAT were assay12, 13.

Polymerase chain reaction

Samples of peripheral blood were taken puncturinq a vein with a syringe and centrifuged for 20 min. The serum samples from patients were frozen and stored at -40ºC until examination. Fifty microliters of a 20% suspension of Chellex-100-iron (Bio-Rad Laboratoriesâ, Hercules, CA) and 1ml of 20 mg/ml of proteinase K was added to 150 ml of plasma. After 15 min. incubation at 65ºC, the samples were boiled in a water bath for 10 min. The Chellex resin was precipitated by centrifugation in an Eppendorf microcentrifuge (Brikman Instruments, Eppendorf, Germany) for 1 min. The supernant was transerred to a new plastic tube; 2.5 ml of which was used for each PCR assay, adjusting the final volume to 25 ml. Written consent was obtained from the human subjects in all cases.

The oligonucleotides used to drive the reaction were derived from L. braziliensis nontranscribed ribosomal gene spacer DNA sequences14 (GenBank accession no M75133). The oligonucleotides used were: forward primer, 5'GCAGCACAGGGAAAG'3; reverse primer, 5'ATGGAGAGAGGCACTAGC-'3. The reaction mixture (final volume, 25 mL) consisted of 50 mM KCI, 10 mM Tris-HCI, pH 8.8, 1.5 mM MgCI2, 1% Triton X-100, 0.2 mM of each of the NTPS, 1 µM of each primer, and 1 unit of Taq polymerase (Promega Corporation, Madison, Wis.) The DNA amplification were done in a thermal cycler (Perkin Elmer Cetus, Norwalk, Conn) programmed as follows: first cycle, 5 min. at 94ºC; second cycle, 1 min. at 60ºC, 1 min. at 72ºC, and 1 min. 94ªC, this cycle being repeated 10 times. The third cycle, 1 min. at 60ºC, 1 min. at 72ºC and 1 min. at 93ºC, was repented 25 times. The fourth cycle consisted of 1 min. at 60ºC and 5 min. al 72ºC. All the reactions presented in this study were conducted simultaneously during a two-day period using the same stocks of reagents and the same equipment and controls. The rest of the supernatant was kept at -70ºC for further assay. To asses the suitability of the sample for PCR, we used a PCR assay targeted on Human ß-globina to produce a 268 bp product; a positive control consisting of a blood sample from a positive patient (L. braziliensis), in whom a primary cutaneous lesion evolved into secondary multiple cutaneous lesions, showing a high degree of metastasis; a negative control with all the components of the reaction mixture but no DNA to check carry over contamination.

The PCR products (5 µL samples) were electrophoresed in 3% agarose gels. The DNA amplifications bands were transferred to nylon filters and hybridized with a digoxigenin-labeled probe. This was prepared as follows: Amplification of recombinant plasmid (pLbbrs4) containing the target sequence was performed with the same primers. The 126-bp amplified DNA band was labeled with digoxigenin by multiprime incorporation of dUTP-digoxigenin using a Genius kit from Boehringer Mannheim. The hybridized probe was detected by chemiluminescence with a Lumi-Phos 530 (Boehringer Mannheim); the exposure time ranged from 5 min to maximum of 20 min (as recommended by the manufacturer).

RESULTS

The Figure 1 shows and ethidium bromide-stained gel containing the results of a PCR on patient serum samples. The lanes 1-5 shows the CL-DNA sample. The lane 7 indicates a strong positive signal the DNA samples in the patient without cutaneous lesion. The serum samples reveals L. braziliensis from Viannia-subgenus-specific 126 bp amplification. Product a band of 126bp was obtained in all positive samples, in general, the signal were clear. To confirm the positive samples, we transferred the gel to a nylon filter and hybridized it with the digoxigenin-labeled probe (see Materials and Methods, data not shown). Human ß- globin was amplified (268 bp band on gel electrophoresis) in all serum including the controls. Negative controls comprising water instead of serum did not show any amplification bands on gel electrophoresis (Lane 6).

Of the 6 patient, 5 of them 3 (60%) female and 2 (40%) male with 2 to 45 years old, reported a severe lymphadenopathy and 2, 15, 27, 30 and 60 cutaneous lesion (mean = 5 x 7 mm to 27 x 35 mm of diameter), with 4 months of evolution, exposed on the body surface: face (cheek, nose, forehead, front), neck, shoulder, hand, forearm and feet. The local reaction to LST was positive in all patients (mean = 26.2 x 28.4 mm of diameter). Of the 5 patients whose lesions were examined histologically, 4 (80%) showed parasites in the tissue sections.

The lesion aspirate and impression smear, were parasitologically positive in 4 cases. The hamsters at post mortem showed disseminated Leishmania infection. Promastigotes were successfully mass cultured. The patient, without cutaneous lesion had a strongly positive; LST of 17 x 23 mm of diameter at 48 hours later and antibodies titers range between 1: 128 to 1: 3200 for the serodiagnosis assay. The patient with 2 cutaneous lesion was parasitologically positive in culture NNN, for serodiagnosis and PCR probes (Table 1).

DISCUSSION

The suitability of the PCR as a diagnostic test for leishmaniosis in endemic area is reported in previously study13. In some works, in patient with L. braziliensis for whom diagnosis is important, the histological confirmation proves most difficult. Impression smears have been reported to be a useful rapid diagnostic,15 whereas the value of immunocytochemistry is disputed.16, 17 The PCR conducted in our laboratory allowed the detection and identification of Leishmania parasite in serum samples from patients with primary lesion and patients without clinical manifestation. The fact that these patients were positive LST, anti-Leshmania antibody test an smear or parasite culture, was indicative that the PCR was detecting true Leishmania infections in all patients.

The PCR yielded an equal or a higher percentage of positive results that were independent of the clinical conditions of the patients. However, the PCR showed a large advantage in the evaluated individuals, because not only increased the sensivity of laboratory diagnosis as the parasitological and immunological techniques, also demonstrated the presence of L. braziliensis in the blood of a patient without clinical symptoms of CL, who remained asymptomatic during the study.

Natural or experimental infections appeared to confer resistance to subsequent leishmanial infection. This immunity was best documented to be a species-specific phenomenon, however, a small number of studies have demonstrated cross protection between some Leishmania species.18 The immunopathological mechanisms probably related with the sequential events, and that could be also responsible for the different clinical aspects found in the man.19 A presumptive diagnosis of leishmaniosis can be made on the basis of the results of laboratory test in conjunction with clinical and epidemiological data. However, the PCR diagnostic test in our study was highly sensitive and allowed the detection of L. braziliensis in all patients.

 

REFERENCES

1.- DAVIES C R, LLANOS-CUENTAS E A, PYKE SDM, DYE C. Cutaneous leishmaniasis in the Peruvian Andes: an epidemiological study of infection and immunity. Epidemiol Infect. 114: 297-318, 1995.        [ Links ]

2.- GRIMALDI G, TESH R. Leishmaniosis of the new world: current concepts and implication for future research. Clin Microbiol Rev 230-250, 1993.        [ Links ]

3.-CACERES-DITTMAR G, TAPIA F J, SANGHEZ M A et al. Determination of the cytokine profile in American cutaneus leishmaniasis using the polymerase chain reaction. Clin Fxp Immunol 91: 500-505. 1993.        [ Links ]

4.- MARDSEN P D, LLANOS-CUENTAS A, LAGO E L, et al. Human mucocutaneous leishmaniasis in Tres Bracos, Bahia, Brazil. An area of Leishmania braziliensis transmission. III. Mucosal disease: presentation and initial evolution. Rev Soc Bras Med Trop 17: 179-186. 1984.         [ Links ]

5.- SARAVIA N G, VALDERRAMA L, LABRADA M, et al. The relationship of Leishmania braziliensis subspecies and immune response to disease expression in New World leishmaniasis. J Infect Dis 159: 725-735, 1989.        [ Links ]

6.- PIRMEZ C. Immunopathology of american cutaneous leishmaniasis. Mem Ins Osw Cruz 87: 105-109, 1992.        [ Links ]

7.- HOWARD J G. Immunological regulation and control of experimental leishmaniasis. Int Rev Exp Parasitol 8: 79, l986.        [ Links ]

8.- STORNI P O, BOLSI F L, YANOWSKY J V. Reación de aglutinación directa para el diagnóstico de la enfermedad de Chagas. Utilización sistemática de 2-Mercapto-ethanol para la eliminación de Aglutininas inespecíficas. Medicina (B. Aires)35: 67, 1975.        [ Links ]

9.- LUGO DE YARBUH A, AÑEZ N, VALERA M, MORENO E. Detección de títulos de anticuerpos, proteínas totales, albúmina y glabulinas en caso de leishmaniasis cutáneas tratados con glucantime. Parasitol Día 20: 20-26, 1996.        [ Links ]

10.- GARDA-MISS M R, ANDRADE-NARVAEZ F J, ESQUIVEL-VIVAS R E et al. Localized cutaneous leishmaniasis (chiclero’s ulcer) in Mexico. Leishmania mexicana mexicana. Trans R. Soc Trop Med Hyg 84:356-358, 1990.        [ Links ]

11.- BRAY R S. GARNHAM P C C. The Giemsa-Colophonium method for staining protozoa in tissue section. Indian J Malar. 16: 153-154. 1962.        [ Links ]

12.- SALINAS G. VALDERRAMA L, PALMA G et Al. Detection of amastigotes in cutaneous and mucocutaneous leishmaniasis using the immunoperoxidase method, using polyclonal antibody sensitivity and specificity compared with conventional diagnosis. Mem Inst Osw Cruz 84: 53-60, 1990.        [ Links ]

13.- PREMOLI-DE-PERCOCO G, LUGO DE YARBUH A, GUEVARA P et al. In situ hybridization of Leishmania braziliensis in tissue. Parasitol al Dia 20: 86-90. 1996.        [ Links ]

14.- GUEVARA P, ROJAS E, GONZALEZ N et al. Presence of Leishmania braziliensis in blood samples of cured patients or at different stages of immunotherapy. Clin Diag Lab Immunol 1: 385-389, 1994.        [ Links ]

15.- BERGER R S, PEREZ-FIGAREDO R A, SPIELVOGEL R L. Leishmaniasis: the touch preparation as a rapid means of diagnosis. J Am Acad Dermatol 16: 1096-1105, 1987.        [ Links ]

16.-SALINAS G, VALDERRAMA L, PALMA G et al. Detection of amastigotes in cutaneous and mucocutaneous leishmaniasis using the immunoperoxidase method, using polyclonal antibody: sensitivity and specificity compared with conventional methods of diagnosis. Mem Inst Osw Cruz 84: 53-60, 1990.        [ Links ]

17.- LUKAS S, KIRKLAM N. Infective granulomas. In Biopsy Pathology of skin, Kirkham N (Editor). London: Chapman and Hall, pp 311-315, 1991.        [ Links ]

18.- MELBY PC. Experimental leishmaniasis in humans. Review Rev Infect Dis 13: 1009-1017, 1991.        [ Links ]

19.- SILVEIRA F T, MORALES M A P, LAINSON R, SHAW JJ. Experimental cutaneous leishmaniasis. 3. Histopathological aspects of the evolution of cutaneous lesion produced in Cebuss-Apella (Primates, Cebidae) by Leishmania (Viannia) lainsoni, I (V) braziliensis and L. (Leishmania) amazonensis. Rev. Inst. Med. Trop de Sao Paulo. 32: 387-394, 1990.         [ Links ]

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