Reliable diagnosis of post-kala-azar dermal leishmaniasis (PKDL) using slit aspirate specimen to avoid invasive sampling procedures


Corresponding Author Poonam Salotra, National Institute of Pathology (ICMR), Safdarjung Hospital Campus, New Delhi 110029, India. Tel.: +91 11 2616 6124; Fax: +91 11 2619 8401; E-mail:



Confirmatory diagnosis of post-kala-azar dermal leishmaniasis (PKDL) is primarily based on invasive skin biopsy procedure. We evaluated the utility of minimally invasive slit aspirate specimen for serological and molecular diagnosis of PKDL. We compared the PKDL diagnosis using slit aspirate and skin biopsy specimens from the same patients.


Serological diagnosis using rK39 strip test was performed with serum and slit aspirate sample; molecular diagnosis for parasite detection and quantification was carried out by quantitative real-time PCR (Q-PCR) with skin biopsy and slit aspirate sample.


The rK39 serological strip test was positive in all PKDL cases with both slit aspirate and serum samples (n = 50) and negative in all control cases (n = 24), giving a sensitivity of 100% (95% CI: 92.9–100%) and a specificity of 100% (95% CI: 86.2–100%). Quantitative-PCR detected parasite in all PKDL slit aspirates (n = 50, sensitivity = 100%, 95% CI: 92.9–100%) and tissue biopsies (n = 46, sensitivity = 100%, 95% CI: 92.3–100; it was negative in all controls including dermal tissues (n = 24) and slit aspirates (n = 24), giving specificity of 100% (95% CI: 86.2–100%). The parasite load in tissue and slit aspirate samples was significantly (P < 0.0001) correlated (r = 0.82).


Slit aspirates are a simpler and minimally invasive sampling technique for initial screening by serology followed by confirmatory diagnosis of PKDL with microscopy and/or Q-PCR. The simplified procedure has the potential for epidemiological studies and assessment of cure in PKDL.



Un diagnostic de confirmation de la leishmaniose dermique post kala-azar (LDPKA) repose principalement des procédures invasives de biopsies cutanées. Nous avons évalué l'utilité de spécimen de ponction d'incision très peu invasive pour le diagnostic sérologique et moléculaire de la LDPKA. Nous avons comparé le diagnostic de la LDPKA utilisant des spécimens de ponctions d'incision et de biopsies cutanées des mêmes patients.


Le diagnostic sérologique à l'aide de bandelettes rk39 a été réalisé avec du sérum et des échantillons de ponction d'incision, le diagnostic moléculaire pour la détection des parasites et la quantification a été réalisée par PCR quantitative en temps réel (Q-PCR) sur des échantillons de biopsie cutanée et de ponction d'incision.


Le test sérologique sur bandelette rk39 a été positive dans tous les cas de LDPKA, à la fois sur les échantillons de ponction d'incision et de sérum (n = 50) et négatif dans tous les cas témoins (n = 24), ce qui donne une sensibilité de 100% (IC95%: 92,9 à 100) et une spécificité de 100% (IC95%: 86,2 à 100). La Q-PCR a détecté des parasites dans toutes les ponctions d'incision de LDPKA (n = 50, sensibilité = 100%, IC95%: 92,9 à 100%) et les biopsies de tissus (n = 46, sensibilité = 100%, IC95%: 92,3 à 100; elle était négative dans tous les cas contrôles, y compris de tissus dermiques (n = 24) et de ponctions d'incision (n = 24), ce qui donne une spécificité de 100% (IC95%: 86,2 à 100%). La charge parasitaire dans les échantillons de tissus et de ponctions d'incision corrélaient significativement (P < 0,0001, r = 0,82).


Les ponctions d'incision sont une technique d’échantillonnage simple et peu invasive pour le dépistage initial par la sérologie suivie d'un diagnostic de confirmation de la LDPKA par la microscopie et/ou la Q-PCR. Cette procédure simplifiée a un potentiel pour les études épidémiologiques et l’évaluation de la guérison dans la LDPKA.



El diagnóstico confirmatorio de la Leishmaniasis cutánea Post Kala-azar (LCPK) está principalmente basado en un procedimiento invasivo de biopsia cutánea. Hemos evaluado la utilidad del aspirado de lesiones abiertas como muestra muy poco invasiva, para el diagnóstico serológico y molecular de LCPK. Hemos comparado el diagnóstico de LCPK utilizando aspirado y muestras de biopsias de piel de los mismos pacientes.


Se realizó un diagnóstico serológico utilizando la tira reactiva rk39 con muestras de suero y muestras de aspirado de lesiones abiertas; diagnóstico molecular para la detección del parásito y cuantificación mediante una PCR cuantitativa a tiempo real (Q-PCR) con biopsia de piel y aspirado de lesiones abiertas.


La prueba con la tira reactiva rk39 era positiva en todos los casos de LCPK tanto con las muestras de aspirado como las muestras de suero (n = 50) y negativa para todos los controles (n = 24), teniendo una sensibilidad del 100% (IC 95%: 92.9–100%) y una especificidad del 100% (IC 95%: 86.2–100%). La Q-PCR detectó parásitos en todas las muestras de aspirados de LCPK (n = 50, Sensibilidad = 100%, IC 95%: 92.9–100%) y biopsias de tejido (n = 46, sensibilidad = 100%, IC 95%: 92.3–100; y era negativa para todos los controles, incluyendo los tejidos cutáneos (n = 24) y aspirados (n = 24), mostrando una especificidad del 100% (IC 95%: 86.2–100%). La carga de parásitos en muestras de tejido y aspirados estaban significativamente correlacionadas (P < 0.0001) (r = 0.82).


Los aspirados de lesiones abiertas son una técnica más simple y muy poco invasiva para realizar un primer cribado mediante serología, seguido por un diagnóstico confirmatorio de LCPK mediante microscopía y / o Q-PCR. El procedimiento simplificado tiene potencial para realizar estudios epidemiológicos y evaluación de la recuperación en LCPK.


The protozoan parasites of the genus Leishmania are the causative agents of a group of diseases called leishmaniasis, endemic in more than 88 countries and affecting 12 million people worldwide. Depending on the species, Leishmania produce a wide spectrum of diseases, from simple cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL) to deadly visceral leishmaniasis (VL). The visceral form of the disease, caused by parasites of L. donovani complex, is prevalent in tropical regions, with more than 90% of total cases reported from India, Bangladesh, Nepal, Brazil and Sudan (Desjeux 2001). Post-kala-azar dermal leishmaniasis (PKDL) is a complication of VL, characterised by macular, maculopapular or nodular rash in a patient who has recovered from VL. In India and Bangladesh, PKDL is reported in 5–10% of patients treated for VL, usually after an interval of a few months to years (Ramesh & Mukherjee 1995; Rahman et al. 2010). By contrast, in Sudan it develops in 50–60% of VL cases within a few weeks of treatment. The reasons of varying incidence of PKDL from country to country are not clear (Zijlstra et al. 2003). The need to search for cases of PKDL and treat them as a part of VL control programme has been emphasised, as patients with PKDL are an important reservoir for the parasite between VL epidemics (Thakur & Kumar 1992).

Diagnosis of PKDL remains a major challenge as the parasite load may be scanty, particularly in macular and maculopapular cases. As a result, it is often misdiagnosed, primarily as leprosy, a co-endemic disease. Diagnosis based on demonstration of parasite by microscopy in slit smear or tissue biopsy is considered the gold standard, but it has the limitation of low sensitivity with variable degree of positivity ranging from 67–100% in nodular lesions, 36–69% in papular lesions and 7–33% in macular lesions (Salotra & Singh 2006). Diagnosis based on immunological methods (direct agglutination test, enzyme-linked immunosorbent assay, rK39 strip test, etc.) is not conclusive because of the persistence of anti-leishmanial antibodies after VL infections (Salotra et al. 2001a). Molecular diagnoses based on PCR (Osman et al. 1998; Salotra et al. 2001b; Mondal et al. 2010), restriction fragment length polymorphism (RFLP) analysis (Schonian et al. 2003) and nested PCR (Sreenivas et al. 2004) are sensitive and specific, but the multiple steps of post-PCR manipulation in these procedures require time and pose the risk of DNA contamination. Recently, quantitative real-time PCR (Q-PCR) was reported as a rapid and accurate method for diagnosis with parasite quantification in various diseases including Toxoplasma gondii (Kompalic-Cristo et al. 2007), Trypanosoma cruzi (Piron et al. 2007), Plasmodium spp. (Perandin et al. 2004) and Leishmania spps (Mary et al. 2004; Aoun et al. 2009; Kumar et al. 2009; Verma et al. 2010). Molecular diagnosis of PKDL is primarily based on DNA isolated from skin biopsy sample, which is invasive, requires technical expertise and carries the risk of bleeding and bacterial or fungal superinfection. As a result, subjects are often reluctant to provide a biopsy sample, which reduces participation rates.

In recent years, studies on molecular diagnosis using simple and non-invasive diagnostic specimens like buccal swabs for VL (Vaish et al. 2011), lesion aspirate for CL (Luz et al. 2009) and filter paper lesion impressions for American CL (Boggild et al. 2010) showed the progress towards non-invasive, rapid and simpler specimens for diagnosis which may facilitate large epidemiological studies. These emphasise the need to develop sensitive diagnostics based on non-invasive, simpler sampling techniques that may be applicable for field application. Earlier, we have shown the utility of nested PCR (Sreenivas et al. 2004) in skin biopsy and slit aspirate specimens for PKDL diagnosis. In this study, we tested the utility of minimally invasive slit aspirate sample for reliable diagnosis of PKDL and assessment of cure.

Materials and methods

Patients and samples

Patients (n = 50) from Bihar who presented at the Department of Dermatology, Safdarjung Hospital, New Delhi, and were diagnosed as PKDL on clinico-histopathological observations, were included in this study. Controls (n = 24) comprised of leprosy, sporotrichosis and pityriasis lichenoides chronica cases. The study was approved by and carried out under the guidelines of the Ethical Committee of the Safdarjung Hospital, India. PKDL cases were treated either with SAG (20 mg/kg/day) for 4 months or with oral miltefosine (150 mg/day) for 2 months, and follow-up samples were collected 1 month after the end of treatment. All patients or responsible adults provided written informed consent for the collection of samples and subsequent analysis.

A fresh blood sample was collected by venipuncture, and serum was isolated. A punch biopsy (4–6 mm) sample was taken under sterile conditions from the lesion part of PKDL and other diseased individuals in NET buffer (150 mm NaCl, 15 mm Tris–HCl pH 8.30 and 1 mm EDTA) for DNA extraction using QIAamp DNA Tissue kit according to manufacturer's instructions. DNA from tissue biopsy was eluted in 100 μl of distilled water.

Skin slit aspiration was carried out by slit scrape technique. The chosen lesion, preferably an indurated one, was cleaned with spirit and allowed to dry. The lesion was gently pinched between index finger and thumb for 1–2 min, exerting enough pressure to blanch it. A clean cut about 5 mm long and 3 mm deep was made with a sterile scalpel to reach the infiltrated layer of dermis. The blade of the scalpel was turned 90°, and using the blunt side of blade, the sides of the cut were scraped two or three times to obtain tissue pulp. This material (approximately 5 μl) was then transferred from scalpel blade to the tube containing 250 μl NET buffer as shown in Figure 1. DNA from slit aspirate was extracted in 25 μl distilled water using QIAamp DNA Tissue kit according to the manufacturer's instructions.

Figure 1.

Slit aspirate collection using slit scrape technique. (a) Cleansing of skin lesion of suspected post-kala-azar dermal leishmaniasis (PKDL) with cotton soaked in spirit, allowed to dry; (b) blanching of lesion by pinching it between index finger and thumb; (c) scraping of the sides of cut with blunt edge of blade to obtain tissue pulp; (d) lesion tissue pulp taken from skin lesion (shown by arrow), transferred to the tube by dipping blade in buffer.

Microscopy in slit smear and tissue biopsy

Slit aspirate was thinly spread on clean glass slide using a circular motion working outwards to avoid damaging any parasites. When the smear was dried, slide was fixed and stained to examine Leishman–Donovan bodies (LDB). Tissue biopsy was taken in a fixative, dehydrated and then embedded in paraffin. The embedded tissue was sectioned into very thin (2–7 μm) sections using a microtome and layered on a glass slide and stained for microscopic examination.

rK39 strip test using slit aspirate and serum sample

The dipstick test using rK39 in the form of antigen-impregnated nitrocellulose paper strips (InBiOS, USA) was performed using 20 μl of patient serum or 20 μl diluted slit aspirate. Three drops of test buffer (PBS + bovine serum albumin) were added to the dried sample. Development of two visible bands indicated the presence of anti-rK39 IgG.

Detection and quantification of parasite load

SYBR Green I-based Leishmania genus-specific real-time PCR was used for detection and accurate quantification of parasite load as described previously (Verma et al. 2010). Briefly, PCR was performed in an ABI Prism 7500 sequence detection system (Applied Biosystems, USA) using forward primer (5′-CTTTTCTGGTCCTCCGGGTAGG-3′) and reverse primer (5′-CCACCCGGCCCTATTTTACACCAA-3′) based on L. donovani kDNA sequence. The PCR was performed in a 10μl sample, consisting of 1X SYBR Green I PCR Master mix (Applied Biosystems), 5 pmol forward primer, 5 pmol reverse primer and 1 μl volume of DNA from the tissue biopsy and slit aspirate sample. The cycling parameters were 50 °C for 2 min, 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All the reactions were performed in triplicate. A standard curve was constructed using 10-fold serially diluted L. donovani parasite DNA corresponding to 103 to 0.1 parasite per reaction. Samples were diluted appropriately to obtain the threshold cycle value (Ct) within the range of standard curve. Results were expressed as the number of Leishmania parasites present in 1μg tissue DNA in case of biopsy and for slit aspirate as the number of Leishmania parasites present in 1 μl slit aspirate.

Statistical analysis

Statistical analysis was performed using Graph Pad Prism 5 software. The differences between experimental groups were analysed using the paired t-test/Mann–Whitney U-test. Correlation was evaluated using Pearson correlation test. All data are presented as mean, and a difference in mean values was considered significant when the P value was <0.05.


Serological diagnosis using slit aspirate and serum samples

The rK39 strip test was positive in all slit aspirate (n = 50) and serum samples (n = 50) of PKDL patients. As slit aspirate used for rK39 strip test was diluted 1 in 50, we attempted rK39 strip test using 1 in 50 diluted serum and obtained positive result in all cases. The test was negative with slit aspirate or serum samples from all controls, comprising of leprosy cases (n = 18) and other skin diseases (n = 6). Representative results with slit aspirate of different categories are shown in Figure 2.

Figure 2.

rK39 strip test. Lanes showing rK39 strip test using slit aspirate as diagnostic specimen: (1) leprosy control; (2) sporotrichosis control; (3) macular post-kala-azar dermal leishmaniasis (PKDL) lesion; (4) papular PKDL lesion; (5) nodular PKDL lesion.

Microscopy results in tissue biopsy and slit smear

Findings of a dense lymphohistiocytic infiltrate beneath an atrophic epidermis, pronounced follicular plugging, vascular hyalinisation and collagen changes in histopathology allowed a suggestive diagnosis of PKDL. The LDB were seen in 15 of 46 (32.6%) tissue biopsy samples (as tissue biopsy was not taken in the three paediatric cases and one adult case refused to give it) comprising mainly of nodular cases (86.7%). The positivity of microscopy with slit aspirate sample was 30 of 50 (60%) slit smears, comprising mainly of nodular cases 66.7% (Table 1).

Table 1. Comparative result of microscopy and Q-PCR with tissue biopsy and slit aspirate samples in different clinical presentations of post-kala-azar dermal leishmaniasis patients
S. no.Age/SexHistory of visceral leishmaniasis (in years)Clinical PresentationLeishman–Donovan bodies in microscopy ofParasite load by Q-PCR in
Tissue biopsySlit aspirateTissue biopsy (Parasites/μg tissue DNA)Slit aspirate (Parasites/μl slit aspirate)
  1. M, Male; F, Female; NA, Biopsy not available; Pos, Positive; Neg, Negative.

328/F10MacularNA NegNA 4
99/F2PapularNA NegNA 13
1012/MNilPapularNA NegNA13
2719/M10NodularPosPos10 307360
3035/MNilNodularNegNeg29 163619
3133/M28NodularNegPos23 000658
3710/M2PapularNA PosNA1364
4522/M13NodularPosPos772314 549
4626/M11NodularPosPos146516 663
4735/M18NodularPosPos46 22818 175
4819/M12NodularPosPos12 94550 312
4932/F6NodularPosPos240 00057 270
5024/M10NodularPosPos590 00070 740

Quantitative-PCR in slit aspirate sample

Q-PCR assay was positive for Leishmania DNA in all slit aspirate samples of PKDL (n = 50), while it was negative in all controls (n = 24), proving utility of slit aspirate as a good diagnostic sample. Further, the assay provided a quantitative estimation of parasite load, indicating a wide range of four to 70 740 parasites/μl slit aspirate, with the mean as 5296 parasites/μl slit aspirate (Table 1). Slit aspirates from nodular cases (n = 26) showed significantly higher (P = 0.0006) parasite load (mean = 9790 parasites/μl slit aspirate) than aspirates from papular/macular cases (n = 24, mean of 427 parasites/μl slit aspirate). Cases where slit smears were LDB positive in microscopy (n = 30) showed significantly (P = 0.015) higher parasite loads (mean = 8205 parasites/μl slit aspirate) than LDB-negative cases (n = 20, mean = 932 parasites/μl slit aspirate). Similarly, cases where the histopathology was LDB positive (n = 15) showed significantly (P = 0.0001) higher parasite load in slit aspirates (mean = 15 925 parasites/μl slit aspirate) than to histologically LDB-negative cases (n = 31, mean = 791 parasites/μl slit aspirate).

To evaluate the utility of slit aspirate for assessment of cure in PKDL, parasite load was evaluated at 1-month post-treatment (n = 19). At the end of treatment, no parasites were detectable in slit aspirate of the majority of cases (17/19, 89.5%); slit aspirates of two patients (no. 43 and 44) showed a residual parasite load of 8 and 7 parasites/μl slit aspirate, respectively. On 1-year follow-up, no parasite was detected in a slit aspirate sample of patient no. 43, while patient no. 44 reported with relapse.

Correlation of parasite load in slit aspirate and tissue biopsy

For comparative evaluation, parasite levels were evaluated by Q-PCR assay in lesion tissue biopsy samples of PKDL patients (n = 46). The mean parasite load in tissue biopsy was 21 855 parasites/μg tissue DNA with range of 2–590 000 parasites/μg tissue DNA. Figure 3 shows the significant (P < 0.0001) correlation (r = 0.82) between parasite load in PKDL lesion biopsy and PKDL lesion slit aspirate. Similar to the slit aspirate results, tissue biopsies from nodular cases (n = 26) showed significantly higher (P < 0.0001) parasite load (mean = 38 205 parasites/μg tissue DNA) than papular/macular cases (n = 20, mean = 599 parasites/μg tissue DNA). Furthermore, cases where tissue biopsies were LDB positive in histopathology (n = 15) showed a significantly (P < 0.0001) higher parasite load (mean = 63 383 parasites/μg tissue DNA) in tissue biopsies than LDB-negative cases (n = 31, mean = 1760 parasites/μg tissue DNA).

Figure 3.

Scatter plot showing parasite load in slit aspirate and tissue biopsy samples from lesions of post-kala-azar dermal leishmaniasis (PKDL) patients. Parasite load in slit aspirate (parasites/μl slit aspirate DNA) and tissue biopsy (parasites/μg tissue DNA) samples was determined by quantitative real-time PCR (Q-PCR).

At 1-month post-treatment stage, parasite load in tissue biopsy was evaluated in 14 cases. Similar to the results with slit aspirate, patient nos. 43 and 44 showed parasite load in tissue biopsy (10 and 8 parasites/μg tissue DNA, respectively) post-treatment.

Sensitivity of microscopy in comparison with parasite load

Comparative analysis of the results of parasite load and direct microscopic examination of slit smear and tissue biopsy showed that microscopy was positive in >80% cases that had parasite load of ≥658 parasites/μl in slit aspirate (Figure 4a) or ≥5515 parasites/μg DNA in tissue biopsy (Figure 4b). The threshold for positivity in tissue microscopy was 502 parasites/μg tissue DNA, as all the cases with parasitaemia below this level were LDB negative, while slit aspirates were microscopically positive even in cases with very low parasite levels (Figure 4).

Figure 4.

Histograms showing distribution of parasite load in slit aspirate (a) and tissue biopsy (b) samples with respect to Leishman–Donovan bodies (LDB) positivity in microscopic examination. The arrows show parasite load value giving more than 80% LDB positivity in microscopy.


Reliable and rapid diagnostic tools are desperately needed to detect PKDL cases, which is necessary for an effective control of VL. Slit aspirate as a diagnostic specimen offers sensitivity and specificity comparable with tissue biopsy with Q-PCR. Furthermore, the slit aspirate-based serological rK39 strip test was positive in all PKDL patients, which shows its utility for initial screening in suspected cases of PKDL.

Tissue biopsy involves cutting to subcutaneous level and suturing the area, often in the face where the lesions generally predominate. As an invasive procedure associated with risks of bleeding and infection, it is not desirable to be repeated on the face. We devised a method of estimating the parasite load in skin slit aspirate, which is far less invasive than biopsy, needs no suturing and leaves a normal skin on healing as there is a simple clean cut. This method is cost-effective as there is no need of anaesthesia, needles and syringes, compared with the highly operator-dependent biopsy. Among different sampling techniques, slit aspirate sampling has the advantages of ease of collection, storage and transportation.

While the performance characteristics of Q-PCR in both diagnostic specimens were comparable, slit aspirate was far less invasive, rapid, easy to perform and well tolerated by patients, making it an attractive diagnostic specimen compared with biopsy. A single-slit-aspirate specimen taken once was adequate for performing microscopy, serology and Q-PCR. The strip test and microscopy with slit aspirate can be performed for an initial screening of PKDL followed by microscopy for confirmatory diagnosis. Cases that are positive in serology but negative in microscopy may be tested in a referral lab by Q-PCR molecular test for confirmation. Such a procedure would simplify the confirmatory diagnosis of PKDL, feasible also in epidemiological studies.

A wide range of parasite burden was observed in dermal biopsy as well as slit aspirate specimens of PKDL patients with nodular lesions showing significantly higher parasite load than macular/papular lesions. Thus, the nodular lesions rich in LDB would play an important role in the disease transmission from human to sand fly. Diagnosis of PKDL by direct microscopy is reported to have low (11–60%) sensitivity (Zijlstra et al. 2000). We made a similar observation in histopathology and slit smear of PKDL lesions with 32.6% and 60% positivity, respectively. As expected, the cases that were LDB positive in microscopy showed significantly (P = 0.015) higher parasite loads in slit aspirates than those that were negative in microscopy. Macular/papular cases showed low parasite loads and were particularly difficult to diagnose by microscopy, with LDB positivity as low as 10% and 41.7%, respectively, similar to the previous reports (Salotra et al. 2003). Our study presents a reliable diagnosis of macular/papular cases with Q-PCR using slit aspirate sample.

Studies on molecular diagnosis of human VL (Vaish et al. 2011) and American CL (Luz et al. 2009; Boggild et al. 2010) showed the importance of simpler and non-invasive methods. Non-invasive methods of obtaining biospecimens are very useful for diagnostics and clinical trials, as well as epidemiological screening studies. Among different sampling techniques, slit aspirate samples have the advantages of ease of collection and storage. Slit aspirate Q-PCR in rK39 strip test positives can be applied widely in endemic areas because of its simplicity, portability and tolerability. Slit aspirate sampling could be promoted for PKDL diagnosis over traditional lesion biopsy.


This work was supported by Indian Council of Medical Research (ICMR), India. S.V. thanks the Council of Scientific and Industrial Research (CSIR), India, for providing a research fellowship. We also express our sincere gratitude to the patients for giving consent to be the part of the study.