Polymerase chain reaction detection of Leishmania DNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Abstract

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detect Leishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmania genus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.

Fig. 1. : spectrum of clinical presentations observed in cutaneous leishmaniasis study patients from Sri Lanka. A: plaque like lesion; B: nodules with a depigmented halo (arrow); C: two volcano-like lesion with depigmentation (arrow); D: wet ulcer; E: dry scaly lesion. All patients presented from the north-central province of Sri Lanka.

Fig. 2. : sensitivity of different polymerase chain reaction (PCR) primer assays. A-C: 10-fold DNA serial dilutions from a Leishmania donovani pure culture were used in PCR reactions with different primer sets as indicated; A: JW 11/12 KDNA PCR (Rodgers et al. 1990) [Lane 1: 100 bp DNA ladder; L2-10: 10-fold descending serial dilutions of Leishmania DNA from 100 ng-1 fg; 11: -ve control (no DNA)]; B: LdF/LdR KDNA PCR (Salotra et al. 2001) [Lane 1: -ve control (no DNA); 2-11: 10-fold ascending serial dilutions ofLeishmania DNA from 0.1 fg-100 ng; 12: 100 bp DNA ladder]; C: LITSR/L5.8S internal transcribed spacer 1 PCR (El Tai et al. 2000) [Lane 1: -ve control (no DNA); 2: 100 bp DNA ladder; 3-11: 10-fold descending serial dilutions of Leishmania DNA from 100 ng-1 fg].

Fig. 3. : detection of Leishmania DNA in punch biopsy samples with selected primers. A: JW11/12 KDNA polymerase chain reaction (PCR) (n = 4/38 samples) [Lane 1: -ve control (no DNA); 2: cutaneous leishmaniasis (CL) sample 3; 3: CL sample 4; 4: CL sample 10; 5: CL sample 11; 6: +ve control (CL culture); 7: 100 bp DNA ladder]; B: LITSR/L5.8S internal transcribed spacer 1 PCR (n = 8/38 samples) [Lane 1:-ve control (no DNA); 2: 100 bp DNA ladder; 3: CL sample 1; 4: CL sample 3; 5: CL sample 5; 6: CL sample 10; 7: CL sample 12; 8: CL sample 13; 9: CL sample 16; 10: CL sample 23; 11: +ve control (CL culture)]; C: PCR negative Giemsa-stained slit skin smear showing rounded amastigote-like forms; D: LdF/LdR KDNA PCR (n = 8/38 samples) [Lane 1: 100 bp DNA ladder; 2: CL sample 1; 3: CL sample 2; 4: CL sample 3; 5: CL sample 4; 6: CL sample 5; 7: CL sample 7; 8: CL sample 8; 9: +ve control (CL culture); 10: -ve control (no DNA)].

Fig. 4. : specificity assays of polymerase chain reaction (PCR). A:Leishmania LdF/LdR KDNA PCR in the presence ofMycobacterium tuberculosis DNA [Lane 1: 100 bp DNA ladder; 2: cutaneous leishmaniasis (CL) sample 35; 3:Leishmania +ve control (CL culture); 4: -ve control (no DNA); 5: CL sample 34; 6: CL sample 37; 7: CL sample 36; 8-10:M. tuberculosis samples (n = 3/5)]; B:Leishmania JW11/12 KDNA PCR in the presence of human, Mycobacterium leprae and M. tuberculosis DNA [Lane 1: -ve control (no DNA); 2: 100 bp DNA ladder; 3: +ve control (CL culture); 4-6: M. lepraeand human DNA samples (n = 3/5); 7-8: M. tuberculosisDNA samples (n = 2/5)]; C: LITSR/L5.8S internal transcribed spacer 1 PCR in the presence of human, M. leprae and M. tuberculosis DNA [Lane 1: -ve control (no DNA); 2: 100 bp DNA ladder; 3-5: M. leprae and human DNA samples (n = 3/5); 6-7: M. tuberculosis DNA samples (n = 2/5); 8: CL sample 32 (repeat); 9: CL sample 36; 10: CL sample 37 (repeat); 11: CL sample 35 (repeat); 12: +ve control (CL culture)].

Fig. 5. : Leishmaniadonovani species-specific restriction fragment length polymorphism- polymerase chain reaction (PCR) assay. Internal transcribed spacer 1 amplified DNA digested withHaeIII {Lane 1: -ve control [no DNA PCR]; 2: 100 bp DNA ladder; 3: +ve control [cutaneous leishmaniasis (CL) culture]; 4: CL sample 1; 5: CL sample 5; 6: CL sample 10}. Samples 5 and 10 were two out of eight samples which were negative using LdF/LdR primers.