Fast, sensitive and specific diagnosis of infections with Leishmania spp. in formalin-fixed, paraffin-embedded skin biopsies by cytochrome b polymerase chain reaction.
BACKGROUND
Northern spread of sandflies and Leishmania spp. has been observed in Europe. Diagnosis can be difficult owing to the various clinical manifestations. Species identification is important for patient management and therapy. Molecular diagnostics is increasingly used for pan-Leishmania detection but species identification remains challenging in formalin-fixed material.


OBJECTIVE
To apply cytochrome b (cytb) polymerase chain reaction (PCR) and sequencing for identification of Leishmania species on formalin-fixed, paraffin-embedded (FFPE) skin biopsies; and to identify species-specific histological patterns.


METHODS
Sixty-nine biopsies (48 patients) diagnosed with leishmaniasis based on the presence of amastigotes in the tissue (n = 41) or granulomatous infiltrates with positive pan-Leishmania real-time PCR (n = 28) were analysed with cytb PCR, sequencing and phylogenetic analysis. Histological sections were analysed; epidemiological data were collected.


RESULTS
Cytb PCR identified Leishmania in all specimens: L. infantum (79%), L. major (8%), L. panamensis (4%), L. tropica (4%), L. killicki (2%) and L. aethiopica (2%). Of the detected species 95% were endemic to the country in which the infection was acquired. Amastigotes were found in 59%. Infiltrates were mainly tuberculoid granulomatous (65%), interstitial (15%) and sarcoidal (10%). Pseudolymphomatous features and pseudocarcinomatous hyperplasia were more common in L. major infections than in L. infantum (P < 0·01).


CONCLUSIONS
Cytb PCR and sequencing is a fast, reliable and sensitive assay for identification of Leishmania spp. in FFPE biopsies. Leishmania infantum is the main cause of cutaneous leishmaniasis in Germany. Tuberculoid granulomas, other types of granulomas and pseudolymphomatous infiltrates may be encountered; the latter being indicative of infection with L. major.
Dermatologikum Hamburg, Stephansplatz 5, 20354, Hamburg, Germany.
M
M
Gebhardt
Dermatologikum Hamburg, Stephansplatz 5, 20354, Hamburg, Germany.
B
B
Ertas
Dermatologikum Hamburg, Stephansplatz 5, 20354, Hamburg, Germany.
T M
TM
Falk
Dermatologikum Hamburg, Stephansplatz 5, 20354, Hamburg, Germany.
N
N
Blödorn-Schlicht
Department of Dermatology, Münster University, Von Esmarch Strasse 58, 48149, Münster, Germany.
D
D
Metze
Dermatologikum Hamburg, Stephansplatz 5, 20354, Hamburg, Germany.
A
A
Böer-Auer
D000293:Adolescent; D000328:Adult; D000368:Aged; D001706:Biopsy; D002648:Child; D002675:Child, Preschool; D045303:Cytochromes b / Q000378:metabolism*; D005260:Female; D006801:Humans; D007891:Leishmania / Q000302:isolation & purification; D016773:Leishmaniasis, Cutaneous / Q000175:diagnosis*; D008297:Male; D008875:Middle Aged; D019095:Point-of-Care Systems; D016133:Polymerase Chain Reaction / Q000379:methods*; D016053:RNA, Protozoan / Q000302:isolation & purification; D055815:Young Adult
D016053:RNA, Protozoan; D045303:Cytochromes b