Advancement in Molecular Diagnosis of Post Kala-Azar Dermal Leishmaniasis.

Post kala-azar dermal leishmaniasis (PKDL), a clinical sequela of visceral leishmaniasis (VL), plays a critical role in the anthroponotic transmission of VL, particularly in the Indian subcontinent (ISC). The early, accurate, and feasible diagnosis of PKDL is essential for the attainment and sustenance of VL elimination goal in ISC. PKDL poses a stumbling block for this goal, considering the heterogeneity presented with regard to time after cure of VL and onset of PKDL, chronicity, and clinical variations. In most of the endemic regions the diagnosis is based on clinical examination, previous history of VL, by ruling out other disorders, and by the response to treatment. The conventional microscopic examination involving the demonstration of Leishman-Donovan bodies (LDB) in macrophages is pathognomonic, however, the method faces constraints in terms of being invasive, less sensitive, technically demanding, and difficult to be applied in field conditions. Serological evidences are of limited use because antileishmanial antibodies remain positive for years after VL treatment. Molecular tools such as PCR, nested-PCR, Q-PCR overcome these constraints and have become increasingly popular due to their high sensitivity and specificity along with their applicability in diverse clinical samples. Molecular methods not only play a key role in early detection but also provide quantification and monitoring of treatment effectiveness. NCBI PubMed search tool was used for locating, selecting, and extracting research articles pertinent for this review article by using various related terminologies on the molecular diagnosis of leishmaniasis. Copyright:

Introduction

Leishmaniasis is a major public health concern globally, which has been recognized as a high priority disease by the World Health Organization.[1] The disease, amongst all parasitic infections, accounts for the third most common cause of mortality after malaria and schistosomiasis.[2] The spectrum of infection ranges from sub-clinical (oligosymptomatic - serologically positive cases with nonspecific clinical manifestations),[3] localized cutaneous leishmaniasis to disseminated forms, mucocutaneous and visceral leishmaniasis (VL). VL, also known as kala-azar, caused by Leishmania donovani complex, is fatal if left untreated. The annual incidence of the disease is 50,000 to 90,000 new cases worldwide[4] resulting in high morbidity and mortality in developing countries.

Post kala-azar dermal leishmaniasis (PKDL), a dermal sequela of VL, is usually seen in patients who are apparently cured of VL in 5%–15% cases in the Indian subcontinent (ISC) against 50% cases in East Africa. PKDL has been reported to occur after an interval of a few months to several years after cure from VL in ISC,[56] as compared to Sudan where it may occur within 0–6 months after successful treatment of VL.[7] In India, in a clinico-epidemiological study, 70% of cases manifested PKDL within 5 years following cure from VL.[6] Similar results showing the occurrence of up to 36% cases of PKDL within 1 year after VL have also been reported.[8] The figures are more alarming in Sudan where approximately 60% of cases manifest PKDL within 0–13 months postVL.[9] The disease may even occur in the absence of the past history of VL.[61011]

PKDL was first described in ISC by Brahmachari[12] in 1922 as “dermal leishmanoid” as the L. donovani bodies had been observed in the lesions, later it was renamed as post kala-azar dermal leishmaniasis due to its appearance postVL (kala azar). In Sudan, PKDL was first explained by Christopherson in 1921.[13] The clinical manifestations in Indian PKDL may include monomorphic macular form predominantly presenting with hypopigmented lesions in macular cases, papulo-nodular cases presenting papular/nodular lesions, mixed, or polymorphic cases with all the three types of lesions [Figure 1]. Other unusual presentations may include erythrodermic, fibroid type, and plaque. No standard system has been proposed for grading severity of Indian PKDL. The severity is usually described as mild (very few lesions), moderate (generalized and easily visible lesions), or severe (dense coverage of body with lesions). In Sudanese PKDL, lesions typically start on the face as papules and spread to other parts of the body. The papules may increase in size turning into nodules or plaques or a combination of these. The combined maculo-papular rash is commonly seen; micropapular rashes resembling measles may also occur.[14] In Indian PKDL, the nodules may coalesce to form large plaques and rarely ulcerate, however, ulceration is common in Sudanese PKDL. Lymphadenopathy and nerve involvement are usual features of Sudanese PKDL but are seldom seen in Indian PKDL. Furthermore, treatment is always required in Indian PKDL, however Sudanese PKDL is mostly self-healing.[7] The patients in which VL and PKDL occur simultaneously are called as Para-PKDL; uncommon in Indian PKDL, but is seen in approximately 16% cases of Sudanese PKDL.[15] The countries afflicted by VL have signed the London Declaration on Neglected Tropical Diseases and have committed to eliminate VL as a public health problem by 2020.[16] Further, India, Nepal, and Bangladesh have targeted at maintaining the annual incidence rate of VL case to <1 per 10,000 population by 2020 at block (India), Upazila (Bangladesh), and district level (Nepal).[17] Owing, to the fact that transmission of VL in ISC is anthroponotic, PKDL cases play a pivotal role being the proposed disease reservoir, especially during inter-epidemic periods of VL. Accordingly, the identification and elimination of PKDL should be an indispensable component of the ongoing VL elimination program. The success story of the elimination campaign in India is proven by the status that 88% of blocks have achieved the elimination target.[1819] PKDL remains a challenge for researchers and clinicians because of its poorly investigated burden and pathogenesis. The present review gives an overview of conventional diagnostic practices and highlights the advancements in molecular tools for the diagnosis of PKDL.

Figure 1

Different clinical presentations of post kala-azar dermal leishmaniasis (PKDL). (a) Hypopigmented macular lesions. (b) Papular lesions predominantly present around mouth and chin. (c) Papulonodular lesions covering the entire face

The data in the review has been summarized from the research articles identified through PubMed search using terminologies, “Post kala-azar dermal leishmaniasis”, “diagnosis + Post kala-azar dermal leishmaniasis/Leishmaniasis”, “PCR + Leishmaniasis” “Loop mediated isothermal amplification for leishmaniasis”. Additional information was extracted from the reference lists of the selected articles. References were selected on the basis of their scientific relevance pertaining to specifically molecular diagnosis of PKDL.

Impediments in the diagnosis of PKDL

The diagnosis of PKDL is primarily based on clinical, epidemiological, and laboratory data. Identification of PKDL is crucial, however, the diagnosis of PKDL remains a big challenge for clinicians especially the macular variant with scanty parasite load. PKDL generally mimics leprosy, often with multi-bacillary forms of leprosy, most commonly with lepromatous leprosy, sometimes with borderline lepromatous, and rarely with mid-borderline leprosy. However, the preservation of sensation demarcates the PKDL lesions from leprosy. If papules are localized on the face, the possibility of rosacea and photodermatoses should be taken into account. The main differential diagnosis for the macular form is pityriasis versicolor, vitiligo, fungal infections, and all forms of leprosy.[142021] Various studies have assessed the sensitivity and specificity of existing diagnostic tools for PKDL but have failed to provide an estimate for the sensitivity of macular variant of PKDL.[22] A report of approximately 27% of cases of PKDL being initially misdiagnosed at primary health centers further raises concern. The majority (78%) of these cases were misdiagnosed as leprosy and received either complete or partial treatment for it.[6] In the last few years, pictorial manuals have been released to enable health workers in the differential diagnosis of PKDL from other skin diseases.[14] It has been documented that 25% of patients seek medical treatment majorly for cosmetic reasons, highlighting the lack of urgency felt by the patients to report early.[23] Recently, it has been reported that shortening the time from health care seeking to the diagnosis could bring about a substantial reduction in the incidence of VL as PKDL cases are carriers of L. donovani.[24] A clinical algorithm has been defined by WHO, which has taken into consideration the regional variations and current control strategies.[14]

Conventional Diagnostic Methods

From the first description of PKDL till date numerous methods have been employed for detection of PKDL, however, the diagnosis is still largely based on clinical examination, the past history of VL, the type of skin lesion and exclusion of other differential dermal manifestations. The various other available methods are discussed below.

Microscopy

Microscopy involving demonstration of Leishman–Donovan bodies (LDB) amastigotes in tissue or slit skin smear is considered to be the gold standard owing to its high specificity. However, this method is invasive, has poor sensitivity involving prolonged searches, and is difficult to be applied in the field. The variable degree of positivity has been reported ranging from 67%–100% in nodular, 36%–69% in papular lesions, and 7%–33% in macular lesions.[25] Further, the culture-based methods involving the isolation of parasites are often not positive and prone to contamination.

Serological methods

Various serological methods, such as enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody test (IFAT), indirect hemagglutination assay (IHA), and immunoblotting have been employed but are of limited value in endemic settings with variable results. The cloning and characterization of several genes of Leishmania has further improvised the serological methods for the diagnosis of VL and PKDL. These include rK39, rKE-16, ORF F, rH2a, rH2b, rGBP, rLACK, rgp63, rP20, rPSA, and purified LPG, etc.[26272829] Of these, two recombinant antigens are commercially used, which are rK39 (from L. chagasi) and rKE-16 (from L. donovani). These antigens have been used in various formats such as ELISA, latex agglutination tests, flow through, and lateral flow rapid tests for the diagnosis of VL and PKDL. The diagnostic potential of recombinant Lepp12 antigen has been evaluated both for VL and PKDL where the majority of PKDL patients were seronegative using this antigen, whereas 100% of VL patients were diagnosed with rLepp12-based western blot.[30]

Direct agglutination test (DAT)

Direct agglutination test (DAT), though has high sensitivity and specificity for the diagnosis of PKDL poses limitations in terms of complex procedure and antigen variability. The sensitivity amongst various studies for DAT varied between 94%–100% and specificity between 40%–100%.[22]

Western blot

Amongst the most specific and sensitive techniques is western blot, which provides information about the parasites' antigenic profile. The western blot analysis based on IgG reactivity differentiated patients with PKDL from others by detection of polypeptides of 67, 72, and 120 kDa. Intrinsic differences have been implicated in the antibodies generated in the sera of patients with VL and PKDL. The high levels of IgG, IgG1, IgG2, and IgG3 antibodies differentiated PKDL cases from those cured of VL. The absence of IgE and IgG4 in patients with PKDL distinguished them from patients with active VL.[31]

Rapid diagnostic tests (RDTs)

No specific and practical serological test exists for PKDL, since a positive antibody test in a suspected case may be attributed to the persistence of past antileishmanial antibodies. The rK39 RDT based on recombinant rK39 is widely used for the detection of both VL and PKDL. It has yielded an exemplary cent percent sensitivity and specificity using both slit aspirate and serum samples of PKDL.[32] The strip is still routinely used in all suspected cases of PKDL along with those with a past history of VL.[33] Despite, being highly sensitive, the rK39 RDT cannot be used as a confirmatory tool for PKDL. Moreover, the absence of VL history in 10%–20% cases is suggestive of sub-clinical infection and poses a hindrance in diagnosis.

Attempts have been made to use a patient-friendly, minimally invasive sampling method for the diagnosis of PKDL.[32] The less invasive approach of using slit-skin smear examination has been reported to be more sensitive than tissue biopsy with a sensitivity of 66% as opposed to 32%–36% in histopathology.[34] Some recently reported noninvasive diagnostic tests employed for diagnosis of VL and PKDL using urine sample include ELISA and dipstick test based on recombinant glycoprotein 63 (rGP63) and recombinant cysteine protease A (rCPA). The urine ELISA gave a sensitivity and specificity of 97.94% and 100%, respectively, for diagnosis of VL, whereas the dipstick test yielded both 100% sensitivity and specificity for VL diagnosis. For PKDL diagnosis both urine ELISA and dipstick gave cent percent sensitivity and specificity.[35] Recent studies assessing various Leishmania membrane proteins [elongation factor 1 (EF1-α), α-tubulin, and glycoprotein 63] as noninvasive diagnostic candidates using VL urine sample revealed good reactivity with all.[36]

Molecular Methods

The drawbacks of histopathological and serological methods have paved the way for molecular methods that are becoming increasingly significant owing to their excellent sensitivity and specificity. Numerous nucleic acid detection methods targeting both DNA and RNA have been developed. The targets employed for the diagnosis of VL are equally applicable for the detection of PKDL cases since the causative organism is L. donovani for both. The molecular-based methods are the only approach to differentially diagnose the cases of PKDL with a past history of VL infection. Various molecular methods employed have been discussed below.

Polymerase chain reaction (PCR)

The PCR-based method provides a powerful tool for the diagnosis of leishmaniasis.[373839] This method has several advantages over the conventional methods, having high sensitivity, rapidity, and being applicable for the identification of species/strain in different clinical specimens.[40] Many DNA targets have been documented for Leishmania such as 18S ribosomal RNA (rRNA), small subunit (SSU) rRNA, a repetitive genomic sequence of DNA, the mini-exon gene repeat, the α-tubulin gene region, gp63 gene locus, internal transcribed spacer (ITS) regions, and microsatellite DNAs, such as maxi- and minicircles of kinetoplast DNA (kDNA). The most widely used target for PCR is the multicopy kDNA as it provides high sensitivity. The nested PCR further enhances the sensitivity and permits a reliable diagnosis of PKDL in a less invasive manner. A nested PCR assay for detecting PKDL using slit aspirate yielded a remarkable positivity of 93% (27/29), which was only 69% (20/29) in primary PCR assay. The assay depicted high sensitivity even in macular cases with low parasite load.[41] Similar types of observations have been reported by others also.[3742] A recent study has reported the sensitivity of primary PCR as 65.4% (17/26), which increased to 88.5% (23/26) in nested PCR in PKDL cases.[33] It has also been reported that PCR has the potential of detecting parasitemia a few weeks before any clinical signs or symptoms appear.[43] Detection of Leishmania DNA from the peripheral blood buffy coat especially from hypopigmented macular lesions has led to the diagnosis of 40%–75% clinically suspected PKDL individuals.[42] The sensitivity in the diagnosis of PKDL by PCR using different targets and clinical specimen ranges between 76%–100%, with 100% specificity in all [Table 1].

Table 1

Sensitivity and specificity of PCR, based on different targets for diagnosis of Post kala-azar dermal leishmaniasis

Target	Sample	Sensitivity	Specificity	Reference
medRNA (mini-exon- derived RNA)	Skin biopsy	100%	100%	[44]
SSUrRNA (small subunit rRNA)	Slit skin smear	83%	100%	[37]
SSUrRNA (small subunit rRNA)	Lymph node aspirate	82%	100%	[37]
kinetoplast DNA (kDNA)	Skin biopsy	94%	100%	[45]
kinetoplast DNA (kDNA)	Skin biopsy	96%	100%`	[39]
kinetoplast DNA (kDNA)	Slit skin smear	93%	100%	[41]
heat shock protein (Hsp) 70 gene	Skin biopsy	100%	100%	[46]
18S ribosomal RNA (rRNA)	Skin biopsy	76%	100%	[47]
kinetoplast DNA (kDNA)	Skin biopsy	94.5%	100%	[48]
Internal transcribed spacer (ITS) region	Skin biopsy	91.9%	100%	[49]
kinetoplast DNA (kDNA)	Slit skin smear	88.5%	100%	[33]

Real-time PCR (Q-PCR)

Real-time PCR has been shown to be superior to the conventional PCR for the diagnosis of leishmaniasis. It has become increasingly popular due to its high sensitivity, quantification, automation, high throughput possibility, and rapidity. Furthermore, it has the applicability on a wide range of samples along with a drastic reduction in the risk of cross-contamination since there is no need to open the tubes for postPCR analysis. It is widely used in referral labs. It can also be utilized for genotyping purpose by using high-resolution melt (HRM) analysis and can be used to differentiate amplicons on the basis of sequence variations. Using the same nucleotide sequence as for conventional PCR, the Q-PCR based on the analysis of fluorescent signal produced either by using intercalating fluorescent dyes SYBR green or fluorescent probe as TaqMan, fluorescence resonance energy transfer (FRET), or minor groove binder (MGB).[50]

Various studies have reported using kDNA as a target for Q-PCR for the diagnosis of VL and for monitoring parasite kinetics. kDNA target in L. infantum provided the highest limit of detection of up to 5 × 104 parasites per PCR reaction tube, allowing detection of less than 1 parasite/mL of blood (0.0125 parasites/ml or 12.5 parasites/μl of blood).[51] Cent percent sensitivity has been reported for kDNA-based Q-PCR in the detection of VL using peripheral blood. The number of parasite genomes per milliliter of blood correlated well with a respective splenic score.[5253] Other kDNA-based Q-PCR have shown the detection of up to 1fg DNA corresponding to 0.001 parasite per reaction with applicability both in VL and PKDL diagnosis.[54] Amongst targets on chromosomal DNA, different regions of ribosomal RNA (rRNA) genes termed ribosomal DNA (rDNA) have been used. The 18S rRNA region is commonly used owing to its conserved region to design primers for detection of Leishmania. By using the 18S rRNA sequences various Leishmania species can be differentiated using melt curve analysis.[55] The ITS regions with variable sequences are employed for species typing. Many protein-coding genes have also been used as a target in Q-PCR assays, such as heat shock protein 70 (HSP70), DNA polymerase, glucose-6-phosphate dehydrogenase (G6PD), glucose phosphate isomerase (GPI), mannose phosphate isomerase (MPI), 6-phosphogluconate dehydrogenase (6PGD), tryparedoxin peroxidase, etc.[50] The sensitivity of Q-PCR for the diagnosis of PKDL ranges between 91%–100% using different targets and clinical specimen and 100% specificity in all [Table 2].

Table 2

Sensitivity and specificity of Real-time PCR (Q-PCR) based on different targets for diagnosis of Post kala-azar dermal leishmaniasis

Target	Sample	Sensitivity	Specificity	Reference
kinetoplast DNA (kDNA)	Skin biopsy	100%	100%	[32]
kinetoplast DNA (kDNA)	Slit skin smear	100%	100%	[32]
Leishmania infantum repeat region	Skin biopsy	100%	100%	[56]
REPL repeats (L42486.1)	Skin biopsy	85%	100%	[57]
kDNA	Skin biopsy	100%	100%	[58]
REPL repeats (L42486.1)	Skin biopsy	91.2%	100%	[59]

Further, Q-PCR has demonstrated a promising potential to be used as a tool for the assessment of cure as highlighted by different studies.[3258] Q-PCR can indicate a complete cure or early detection of treatment failure or relapse. The recent study on the monitoring of parasite kinetics using Q-PCR in PKDL patients treated with miltefosine and liposomal amphotericin-B (LAmB), showed a resurgence of parasite in some patients treated with LAmB 6 months posttreatment indicative of treatment inadequacy.[58] Despite the remarkable progress made by the advent of Q-PCR, it is still far from routine clinical application due to the high cost of equipment and reagents, acting as a hitch in its employment in field conditions.

Loop-mediated isothermal amplification (LAMP)

Loop-mediated isothermal amplification (LAMP) is anticipated to be an innovative and novel technique to amplify DNA with high specificity and rapidity under isothermal conditions in the presence of Bst DNA polymerase, which possesses unique strand displacing activity.[606162] It does not require any sophisticated instrument or complicated analysis. The method uses four to six different primers specifically designed to recognize six to eight sequences. Moreover, the sensitivity is less affected by the inhibitory components present in DNA samples.[6364] Numerous studies have successfully established the potential of LAMP for the diagnosis of leishmaniasis.[6265666768697071727374] A recently developed SYBR green I closed tube LAMP assay has depicted exemplary sensitivity (97%) and specificity (100%) for diagnosis of both VL and PKDL. The assay detects various species of Leishmania with the highest sensitivity for L. donovani (1fg), followed by L. tropica (1 pg) and L. major (100 pg).[6566] Also, the assay has shown potential application for the assessment of cure at posttreatment stages, where 4 patients were positive and on longitudinal follow up 2 returned with relapse.[66] Furthermore, strengthening its utility for the mass surveillance of leishmaniasis, the assay has been successfully validated at two endemic sites in India.[68] This assay has already been taken up by industry to develop it in a kit format for employing it in field conditions for mass screening of Leishmania infection in endemic regions.

The use of direct crude clinical samples instead of extracted DNA has also been explored in order to surpass the need for DNA isolation, making the assay both cost and time effective. The use of LAMP based on direct-blood-lysis yielded promising sensitivity of 93.06% for the diagnosis of VL.[67] On similar lines, the direct sample lysis using slit aspirate in the case of PKDL may be explored and developed as a less invasive LAMP assay.

Concluding Remarks

PKDL is a baffling and stigmatizing disease carrying a substantial socioeconomic burden, which is further magnified by the reluctance to obtain treatment or noncompliance. Theoretically, the existence of even a single case of PKDL can be a risk for a new outbreak of VL.[75] Thus, active surveillance, diagnosis, and effective standard treatment of PKDL are critical for the sustenance of VL elimination in ISC. A combination of clinical examination along with serological and molecular methods employed in referral laboratories is usually not feasible in endemic field settings [Figure 2].

Figure 2

Approach adopted for diagnosis of post kala-azar dermal leishmaniasis (PKDL) at a referral center

Molecular methods overcome the constraints of conventional methods as they detect active infection and may also be employed as a tool for assessment of cure. However, these methods employ expensive reagents, sophisticated instruments, complex post-PCR analysis and hence are limited to referral hospitals and research centers. The advent of isothermal techniques has addressed these issues by yielding excellent sensitivity and specificity, being highly time and cost-efficient and applicable in field conditions.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.