BACKGROUND: Cutaneous leishmaniasis is endemic in the Middle East and North Africa. Confirming the diagnosis histologically depends on amastigote identification, which varies significantly depending on the inoculum, strain type, host response and disease stage. Accurate histological diagnosis is mandatory for appropriate therapy. METHODS: Skin biopsies from 122 patients from Lebanon, Syria and Saudi Arabia with clinical diagnosis of untreated leishmaniasis were reviewed and clinical data extracted. Cases were classified according to the modified Ridley's parasitic index. DNA was extracted from formalin-fixed paraffin-embedded blocks. Polymerase chain reaction (PCR) was performed using Leishmania-specific ribosomal internal transcribed spacer 1 (ITS1-PCR). Nested ITS1-PCR was performed on cases negative for conventional ITS1-PCR. ITS1-PCR amplicons were digested with HaeIII for subsequent restriction fragment length polymorphism (RFLP) subspeciation. RESULTS: Of 122 cases, 54 (44.3%) showed a parasitic index of 0-1+ (no unequivocal amastigotes). ITS1-PCR (conventional and nested) was positive for all cases as compared with negative control tissue. RFLP identified Leishmania tropica in all cases. Patients with clinically suspected leishmaniasis, whose skin biopsies failed to detect amastigotes represented 44.3% of our cases. CONCLUSIONS: In this study, we describe a rapid and optimized protocol from DNA extraction to leishmaniasis subspeciation. ITS1-PCR showed high sensitivity and specificity in confirming clinically suspected cases.
BACKGROUND:Cutaneous leishmaniasis is endemic in the Middle East and North Africa. Confirming the diagnosis histologically depends on amastigote identification, which varies significantly depending on the inoculum, strain type, host response and disease stage. Accurate histological diagnosis is mandatory for appropriate therapy. METHODS: Skin biopsies from 122 patients from Lebanon, Syria and Saudi Arabia with clinical diagnosis of untreated leishmaniasis were reviewed and clinical data extracted. Cases were classified according to the modified Ridley's parasitic index. DNA was extracted from formalin-fixed paraffin-embedded blocks. Polymerase chain reaction (PCR) was performed using Leishmania-specific ribosomal internal transcribed spacer 1 (ITS1-PCR). Nested ITS1-PCR was performed on cases negative for conventional ITS1-PCR. ITS1-PCR amplicons were digested with HaeIII for subsequent restriction fragment length polymorphism (RFLP) subspeciation. RESULTS: Of 122 cases, 54 (44.3%) showed a parasitic index of 0-1+ (no unequivocal amastigotes). ITS1-PCR (conventional and nested) was positive for all cases as compared with negative control tissue. RFLP identified Leishmania tropica in all cases. Patients with clinically suspected leishmaniasis, whose skin biopsies failed to detect amastigotes represented 44.3% of our cases. CONCLUSIONS: In this study, we describe a rapid and optimized protocol from DNA extraction to leishmaniasis subspeciation. ITS1-PCR showed high sensitivity and specificity in confirming clinically suspected cases.
Authors: Mario Saab; Hussein El Hage; Khalil Charafeddine; Robert H Habib; Ibrahim Khalifeh Journal: Am J Trop Med Hyg Date: 2015-01-05 Impact factor: 2.345
Authors: Marcos E de Almeida; Ozgur Koru; Francis Steurer; Barbara L Herwaldt; Alexandre J da Silva Journal: J Clin Microbiol Date: 2016-12-28 Impact factor: 5.948
Authors: Milena de Paiva-Cavalcanti; Rayana Carla Silva de Morais; Rômulo Pessoa-E-Silva; Lays Adrianne Mendonça Trajano-Silva; Suênia da Cunha Gonçalves-de-Albuquerque; Diego de Hollanda Cavalcanti Tavares; Maria Carolina Accioly Brelaz-de-Castro; Rafael de Freitas E Silva; Valéria Rêgo Alves Pereira Journal: Cell Biosci Date: 2015-06-17 Impact factor: 7.133