The relevance of characterizing Leishmania from cutaneous lesions: a simple approach for isolation

Barral, Aldina; Almeida, Roque Pacheco de; Jesus, Amélia Ribeiro de; Neto, Eduardo Medeiros; Santos, Ilma; Johnson, Warren D.

doi:10.1590/s0074-02761987000400018

Cited by 15 publications

(7 citation statements)

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“…These results are consistent with other ATL studies from Brazil [4], [27], [28] and other tropical countries. In fact, in Minas Gerais State, government records show the majority of cases to be LCL (95%) followed by MCL (3%) [29].…”

Section: Discussionsupporting

confidence: 93%

American Tegumentary Leishmaniasis: Effectiveness of an Immunohistochemical Protocol for the Detection of Leishmania in Skin

et al. 2013

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BackgroundAmerican tegumentary leishmaniasis (ATL) is endemic in Latin America, where Brazil has over 27 thousand cases per year. The aim of the present study was to develop an immunohistochemical method (IHC) for ATL diagnosis. For this purpose, we used serum from a dog naturally infected with Leishmania (L) infantum (canine hyperimmune serum) as the primary antibody, followed by a detection system with a secondary biotinylated antibody.MethodologySkin samples were obtained from 73 patients in an endemic area of Caratinga, Minas Gerais (MG) State, Brazil all testing positive for ATL with the Montenegro skin test, microscopy, and PCR. Canine hyperimmune serum of a dog naturally infected with Leishmania (L.) infantum was employed as a primary antibody in an immunohistochemical diagnostic method using streptavidin-biotin peroxidase. To assess the specificity of this reaction, IHC assays employing two monoclonal antibodies were carried out. As the polymer-based technology is less time-consuming and labor intensive than the IHC labeled streptavidin-biotin peroxidase method, we compared the two methods for all samples.ResultsThe IHC method detected ATL in 67 of the 73 cases (91.8%). Immunolabeled parasites were primarily detected inside macrophages either in the superficial or the deep dermis. Detection was facilitated by the high contrast staining of amastigotes (dark brown) against the light blue background. A lower detection rate (71.2%) was observed with the both of the monoclonal Leishmania antibodies compared to the canine hyperimmune serum. This may have been due to a non-specific background staining observed in all histological samples rendering positive detection more difficult. The higher efficacy of the canine hyperimmune serum in the IHC method was confirmed by the method using streptavidin-biotin peroxidase as well as that with the polymer-based technology (biotin-avidin-free system).ConclusionsThe data are encouraging with regard to validating IHC as a standard alternative method for ATL diagnosis.

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Section: Discussionsupporting

confidence: 93%

American Tegumentary Leishmaniasis: Effectiveness of an Immunohistochemical Protocol for the Detection of Leishmania in Skin

et al. 2013

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“…However, this is not always possible, since L.(V.) braziliensis grows poorly in culture media, infects experimental animals with difficulty and appears in small numbers in smears and histological sections obtained from the lesions (Marsden 1986, Barral et al 1987. Because it is difficult to find parasites in active lesions (Marsden 1986), indirect ATL diagnosis is based on epidemiological evidence as well as clinical aspects, MST, serology and histopathological examination.…”

Section: Discussionmentioning

confidence: 99%

Leishmanial antigens in the diagnosis of active lesions and ancient scars of American tegumentary leishmaniasis patients

Schubach

Cuzzi-Maya

Oliveira

et al. 2001

Mem. Inst. Oswaldo Cruz

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Cutaneous biopsies (n = 94) obtained from 88 patients with American tegumentary leishmaniasis were studied by conventional and immunohistochemical techniques. Specimens were distributed as active lesions of cutaneous leishmaniasis (n = 53) (Group I), cicatricial lesions of cutaneous leishmaniasis (n = 35) (Group II) and suggestive scars of healed mucosal leishmaniasis patients (n = 6) (Group III). In addition, active cutaneous lesions of other etiology (n = 24) (Group C1) and cutaneous scars not related to leishmaniasis (n = 10) (Group C2) were also included in the protocol. Amastigotes in

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“…Reports of prospective studies to assure sensitivity of culture for the diagnosis of cutaneous leishmaniasis frequently involve patients with infections due to diverse species, disease acquired at different geographic regions and a wide variety of clinical forms (Weigle et al 1987, Barral et al 1987. Problems arise when these results are used to guide diagnostic interventions in areas with transmission of other species and different clinical presentation.…”

Section: Discussionmentioning

confidence: 99%

Sensitivity of a Vacuum Aspiratory Culture Technique for Diagnosis of Localized Cutaneous Leishmaniasis in an Endemic Area of Leishmania (Viannia) braziliensis Transmission

Romero

Sampaio

Macêdo

et al. 1999

Mem. Inst. Oswaldo Cruz

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Sixty eight patients with localized cutaneous leishmaniasis from an area with Leishmania (Viannia) Culture sensitivity for diagnosis of cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis (L.V.b.) has shown variable results depending on quality of culture media (Shaw & Lainson 1981, Cuba et al. 1986), collection technique (Evans 1989) and other characteristics such as disease duration and previous use of anti-leishmanial drugs (Cuba et al. 1984). Marzochi et al. (1993) described a new collection technique using direct inoculation of culture tubes containing NNN media with a liquid phase through a skin puncture system with five to ten mililiters vacuum.We modified the culture medium using a smaller amount of an unenriched liquid phase composed of isotonic saline solution with gentamicin, resuming 15 ml vacuum in each tube. We used the same aspiratory puncture system with commercial needles but the aspiration was modified by allowing air to come into the tubes when pulling the needle out to obtain a larger amount of inoculum. We studied the sensitivity of this method for diagnosis of cutaneous leishmaniasis in a group of pa- (Llanos-Cuentas et al. 1984, Rosa et al. 1988. MATERIALS AND METHODSCulture medium was prepared using blood agar base no. 2 (DIFCO cod. 0696-17) with 15% defibrinated rabbit blood which was added after fusion of agar at 50 o C. Gentamicin (100 µg/ml) and 5-fluorocytosine (100 µg/ml) were added and the mixture was distributed in 10 ml glass tubes (Vacutainer R ). Tubes were covered with rubber caps and vacuum restored aspirating 15 ml with a 20 ml syringe using a 22 gauge needle. A liquid phase composed of 0.3 ml isotonic saline with gentamicin (100 µg/ml) was added immediately before the aspiratory puncture by injection through the rubber cap with a 1 ml syringe and a 22 gauge needle. All injection procedures were performed through the rubber cap after desinfecting it with 0.2% iodinated alcohol.The aspiration puncture was made with the commercial Vacutainer R needle holder dispositive and 21 gauge needles for vacuum blood extraction. The puncture site was cleaned with 0.2% iodinated alcohol and local anesthesia was induced with 0.3 ml lidocaine (2%) injected with a 1 ml syringe with a 13 gauge needle. The procedure was performed through intact skin close to the ulcer border at a 20 o angle with a rotatory movement

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The relevance of characterizing Leishmania from cutaneous lesions: a simple approach for isolation

Cited by 15 publications

References 0 publications

American Tegumentary Leishmaniasis: Effectiveness of an Immunohistochemical Protocol for the Detection of Leishmania in Skin

American Tegumentary Leishmaniasis: Effectiveness of an Immunohistochemical Protocol for the Detection of Leishmania in Skin

Leishmanial antigens in the diagnosis of active lesions and ancient scars of American tegumentary leishmaniasis patients

Sensitivity of a Vacuum Aspiratory Culture Technique for Diagnosis of Localized Cutaneous Leishmaniasis in an Endemic Area of Leishmania (Viannia) braziliensis Transmission

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