Nested PCR Assay for Detection of Leishmania donovani in Slit Aspirates from Post-Kala-Azar Dermal Leishmaniasis Lesions

Abstract

A nested PCR assay to detect parasite DNA in slit aspirates from skin lesions of patients with post-kala-azar dermal lesihmaniasis (PKDL) is described. PCR results were positive in 27 of 29 (93%) samples by nested PCR assay, while only 20 of 29 (69%) were positive in a primary PCR assay. The nested PCR assay allowed reliable diagnosis of PKDL in a noninvasive manner.

Post-kala-azar dermal leishmaniasis (PKDL) is an unusual dermatosis that develops as a sequel of kala azar (KA) or visceral leishmaniasis in 10 to 20% of cured cases in India and in 50 to 60% of visceral leishmaniasis cases in Sudan (6, 11). The disease presents as erythema, hypopigmented macules, and combinations of papules, nodules, and plaques. KA transmission in India is thought to be anthroponotic, and in the absence of animal reservoirs, PKDL patients are deemed to be the singular source of the parasites. Therefore, the identification and treatment of PKDL cases are recognized as prerequisites for the successful elimination of KA (10).

Reports on the evaluation of PCR-based diagnostic assays for PKDL are limited (2, 3, 5, 8). We used a kinetoplast DNA minicircle sequence from Leishmania donovani and developed an assay that detected kinetoplast DNA from a single parasite in the presence of a huge excess of human DNA (8). To further augment the sensitivity of the PCR, we developed a nested PCR method that permitted the diagnosis of PKDL in slit aspirate samples.

We included in the study 29 patients at the pretreatment stage who reported to Safdarjung Hospital, New Delhi, and were clinically and histopathologically confirmed as having PKDL (9). Prior consent was obtained for collecting the biopsy material, following the guidelines of the Safdarjung Hospital ethical committee. Of the 29 patients, 21 had polymorphic presentation, while the remaining 8 patients had a predominantly macular presentation. Histopathological findings on skin biopsy samples were similar to those reported earlier (4, 9). To isolate DNA, PKDL lesion slit aspirate samples and skin biopsy samples were collected in NET buffer (150 mM NaCl, 15 mM Tris-HCl [pH 8.30], 1 mM EDTA). DNA from slit aspirates was isolated by using a QIAamp DNA blood minikit (QIAGEN) following the manufacturer's instructions except that proteinase K digestion was carried out for 60 min at 50°C. DNA from skin biopsy samples was extracted by using phenol-chloroform (8).

Nested PCR assay.

The primers for the first-round PCR assay were previously described (8). Nested PCR primers encompassing a 385-bp fragment internal to the 592-bp minicircle sequence (8) had the sequences 5′-TCGGACGTGTGTGGATATGGC-3′ and 5′-CCGATAATATAGTATCTCCCG-3′. All necessary precautions to avoid contamination were taken.

The PCR conditions for the first-round reaction were as previously described (8). For the second round, 1 μl of the diluted (1:10) products from the first-round reaction was used. The reaction mixture (50 μl) contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 200 μM concentrations of each deoxynucleoside triphosphate, 2 μM concentrations of each primer, and 1.25 U of Taq DNA polymerase (Invitrogen). Amplification was performed in a thermal cycler (Perkin Elmer) programmed for 35 cycles of denaturation at 94°C for 60 s, annealing at 49°C for 60 s, and extension at 72°C for 90 s, preceded by an initial denaturation for 4 min at 94°C. Final extension was for 3 min at 72°C.

The nested PCR assay was tested on reference L. donovani isolates (MHOM/IN/83/AG83 and MHOM/IN/80/DD8) and several Leishmania isolates prepared from the PKDL lesion samples. Parasite DNA in all cases yielded the expected 385-bp fragment. Further, the nested PCR primers could amplify from 1fg of total parasite DNA, which is less than the total DNA from a single Leishmania parasite (data not shown). The specificity of the nested PCR primers was also evaluated by using DNA (1 ng) from microorganisms such as Plasmodium falciparum, Mycobacterium leprae, and Mycobacterium tuberculosis that cause common infectious diseases prevalent in India; there was no amplification of the DNA from any of these organisms when the nested PCR primers were used.

The clinical utility of the assay was established by the PCR amplification of DNA extracted from slit aspirates as well as tissue biopsy specimens from a number of PKDL patients along with suitable controls (Fig. 1). On amplification, the nested PCR primers could amplify parasite DNA from a significant number of samples that were negative in the primary amplification reaction with slit aspirates, while DNA from tissue biopsy specimens (27 of 29) gave positive results in both primary and nested PCR assays (Table 1). The difference in the sensitivities of the nested versus the primary PCR amplification of slit aspirates was statistically significant (P < 0.05). Samples of normal dermal tissue from unaffected parts of the skin of PKDL patients (n = 2) and leprosy cases (n = 3) were all negative. The authenticity of primary and nested amplification reactions was confirmed by nucleotide sequence analysis of the PCR products amplified from clinical samples that were found to be identical with the published sequence (GenBank accession no. Y11401).

FIG. 1.

Detection of L. donovani in DNA extracts of clinical samples. First-round PCR with DNA extracted from PKDL lesion biopsy specimen (lane 1); PKDL slit aspirate samples (lanes 2 to 3); normal uninfected skin biopsy specimen of PKDL patient (lane 4). Lanes 5 to 8 represent the second-round amplification with 1 μl of diluted (1:10) products from the first round in the same order. Products were analyzed by electrophoresis in 1% agarose gel containing 0.5 μg of ethidium bromide/ml in TAE buffer (0.04 M Tris acetate, 0.001 M EDTA) and photographed under UV illumination. Lane M, 1-kb ladder.

TABLE 1.

Detection of L. donovani in PKDL patients by culture, histopathology, and PCR

Method and sample source	No. of positive samples/no. of samples tested	% Positive
Culture	15/22	68
Histopathology
Tissue	14/26	54
Slit aspirate	9/24	38
First-round PCR
Tissue	27/29	93
Slit aspirate	20/29	69
Nested PCR
Tissue	27/29	93
Slit aspirate	27/29	93

Histopathological analysis by hematoxylin-eosin staining (4) of the slit aspirate smears revealed in 9 out of 24 cases (37.5%) Leishman-Donovan bodies that appeared as large dark brown spherules, while 14 of 26 (54%) skin biopsy specimens were positive for Leishman-Donovan bodies. In addition, attempts to set up in vitro cultures of the parasite from lesion biopsy material by using a methodology described earlier (9) gave positive results in 15 of the 22 (68%) cases tested (Table 1).

The sensitivity of the nested PCR primers allowed us to amplify minicircle sequences from parasite material obtained from slit aspirate samples of PKDL lesions in a straightforward way, precluding any need for invasive methods of sample recovery that leave disfiguring marks on the face. Some of the ambiguous results in the primary PCR assay were successfully resolved in the ensuing nested reaction.

Parasite detection presents a challenge in PKDL cases with macular presentation because of a low parasite load. Conventional hematoxylin-eosin staining yielded limited success, and the use of Leishmania-specific monoclonal antibodies improved the sensitivity of detection to 72% in macular cases (9). The nested PCR assay used in the present study circumvented such limitations and showed a high degree of sensitivity even in macular cases. Cases of PKDL infection in patients with human immunodeficiency virus (HIV) have been reported (1, 7). Since the incidence of HIV infection is on the increase in India, HIV patients coinfected with Leishmania are likely to present in the future. In such cases the assay described here would have the potential to provide rapid and reliable detection of L. donovani.