Validation of the polymerase chain reaction for the diagnosis of human cutaneous leishmaniasis in north-west Colombia

Abstract

Leishmania species of the subgenus Viannia account for 88% of all cases of leishmaniasis recorded in Colombia. Correct diagnosis is essential as infection with members of this subgenus can produce disfiguring destruction of the mucosa. Several methods are available to diagnose leishmaniasis in clinical samples. More recently, the polymerase chain reaction (PCR) has been used, with varying sensitivities and specificities depending on the primers used. In this paper we report on the sensitivity and specificity of PCR primers B1/B2 used on clinical samples and compare their use to the conventional parasitological methods. PCR alone is more sensitive than any single conventional method used, but a combination of conventional methods produced comparable sensitivity. PCR is well suited for use in selected cases and as a test for mucosal leishmaniasis.

Polymerase chain reaction with lesion scrapping for the diagnosis of human American tegumentary leishmaniasis

“…The PCR using lesion scrapping presented 100% of sensitivity when compared with direct microscopy, according to Belli et al (1998). Isaza et al (2002) have obtained 90.6% of PCR positivity in biopsies and 80.5% in lesion scrapping. Among the ATL (positive PD and/or MST), the results showed 91.1% of positivity, similar to the ones found by Lopez et al (1993), Rodriguez et al (1994, Pirmez et al (1999), Marques et al (2001), and Weigle et al (2002).…”

“…In Brazil, the disease has been found in all states (Ministério da Saúde 2005). The importance of the ATL laboratorial diagnosis is due to the similarity of clinical signs to other diseases, and the need of quickly initiating the adequate treatment (Isaza et al 2002). The ATL diagnosis is commonly performed by parasite direct search in lesion, antibody search, besides the late hypersensitivity reaction (Montenegro skin test) (Ramírez et al 2000, Badaró & Reed 2001, Hepburn 2003, Singh & Sivakumar 2003.…”

“…The decision of using material collected by scrapping from the lesion's internal edge was due to the fact that it is a less invasive and quicker method, and has good positivity in parasite direct search and in PCR (Belli et al 1998, Isaza et al 2002. The PCR using lesion scrapping presented 100% of sensitivity when compared with direct microscopy, according to Belli et al (1998).…”

Polymerase chain reaction with two molecular targets in mucosal leishmaniasis' diagnosis: a validation study

“…Positive control DNA and biopsy samples were immersed to complete lysis with 500 µl of NET 10 solution (NaCl 5M; buffer 10 mM Tris-HCl (pH8.0) and 10 mM EDTA; SDS 1%) and 20 µl of proteinase K (20 mg/ml) and heated at 56°C for 18 h. The phenol-chloroform extraction protocol described by Isaza (2002) was used. The final DNA pellet was dissolved in 50 µl of TE buffer 0.1× and stored at -70°C until used.…”

Definición de las condiciones de temperatura y almacenamiento adecuadas en la detección de ADN de Leishmania por PCR en flebotominos.

“…La incorporación del uso del Chelex 100 en el proceso de extracción de ADN de protozoarios a partir de muestras de pequeños insectos es una alternativa práctica y eficiente frente a otros protocolos (31). La experiencia de Schriefer et al (20) es diferente a la nuestra respecto a la extracción de ADN con Chelex 100, considerando que ellos tuvieron algunas dificultades para detectar infección con parásitos de malaria tanto en mosquitos individuales como en pequeños volúmenes de sangre.…”