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Table of Contents 
IJD® SYMPOSIUM
Year : 2020  |  Volume : 65  |  Issue : 6  |  Page : 465-472
Advancement in molecular diagnosis of post kala-azar dermal leishmaniasis


ICMR- National Institute of Pathology (NIP), New Delhi, India

Date of Web Publication23-Oct-2020

Correspondence Address:
Poonam Salotra
ICMR - National Institute of Pathology (NIP), New Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijd.IJD_311_19

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   Abstract 


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.


Keywords: LAMP, post kala-azar dermal leishmaniasis, rK39 RDT, VL elimination


How to cite this article:
Dixit KK, Singh R, Salotra P. Advancement in molecular diagnosis of post kala-azar dermal leishmaniasis. Indian J Dermatol 2020;65:465-72

How to cite this URL:
Dixit KK, Singh R, Salotra P. Advancement in molecular diagnosis of post kala-azar dermal leishmaniasis. Indian J Dermatol [serial online] 2020 [cited 2020 Nov 24];65:465-72. Available from: https://www.e-ijd.org/text.asp?2020/65/6/465/298968





   Introduction Top


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,[5],[6] 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.[6],[10],[11]

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.[18],[19] 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

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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.[14],[20],[21] 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 Top


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.[26],[27],[28],[29] 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 Top


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.[37],[38],[39] 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.[37],[42] 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

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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.[52],[53] 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

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Further, Q-PCR has demonstrated a promising potential to be used as a tool for the assessment of cure as highlighted by different studies.[32],[58] 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.[60],[61],[62] 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.[63],[64] Numerous studies have successfully established the potential of LAMP for the diagnosis of leishmaniasis.[62],[65],[66],[67],[68],[69],[70],[71],[72],[73],[74] 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).[65],[66] 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 Top


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

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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.

Acknowledgements

The authors are thankful to Dr. V. Ramesh, Department of Dermatology, Vardhaman Mahavir Medical college, Safdarjung Hospital, New Delhi, India for critically reviewing the manuscript.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
WHO Technical Report Series, 2012. Available from: https://www.who.int/neglected_diseases/resources/who_trs_975/en/. [Last accessed on 2020 May 20].  Back to cited text no. 1
    
2.
Pace D. Leishmaniasis. J Infect 2014;69:S10-18.  Back to cited text no. 2
    
3.
Gama MEA, Costa JML, Gomes CMC, Corbett CEP. Subclinical form of the American visceral leishmaniasis. Mem Inst Oswaldo Cruz 2004;99:889-93.  Back to cited text no. 3
    
4.
5.
Rahman KM, Islam S, Rahman MW, Kenah E, Ghaliv CM, Zahid MM, et al . Increasing incidence of Post kala-azar dermal leishmaniasis in a population-based study in Bangladesh. Clin Infect Dis 2010;50:73-6.  Back to cited text no. 5
    
6.
Ramesh V, Kaushal H, Mishra AK, Singh R, Salotra P. Clinico-epidemiological analysis of Post kala-azar dermal leishmaniasis (PKDL) cases in India over last two decades: A hospital based retrospective study. BMC Public Health 2015;15:1092.  Back to cited text no. 6
    
7.
Zijlstra EE, Musa AM, Khalil EAG, el-Hassan IM, el-Hassan AM. Post kala-azar dermal leishmaniasis. Lancet Infect Dis 2003;3:87-98.  Back to cited text no. 7
    
8.
Zijlstra EE, Alves F, Rijal S, Arana B, Alvar J. Post kala-azar dermal leishmaniasis in the Indian subcontinent: A threat to the South-East Asia Region Kala-azar Elimination Programme. PLoS Negl Trop Dis 2017;11:e0005877.  Back to cited text no. 8
    
9.
Musa AM, Khalil EAG, Raheem MA, Zijlstra EE, Ibrahim ME, Elhassan IM, et al. The natural history of Sudanese Post kala-azar dermal leishmaniasis: Clinical, immunological and prognostic features. Ann Trop Med Parasitol 2002;96:765-72.  Back to cited text no. 9
    
10.
Das VNR, Ranjan A, Pandey K, Singh D, Verma N, Das S, et al . Clinical epidemiologic profile of a cohort of Post kala-azar dermal leishmaniasis patients in Bihar, India. Am J Trop Med Hyg 2012;86:959-61.  Back to cited text no. 10
    
11.
Desjeux P, Ghosh RS, Dhalaria P, Strub-Wourgaft N, Zijlstra EE. Report of the post kala-azar dermal leishmaniasis (PKDL) consortium meeting, New Delhi, India, 27–29 June 2012. Parasit Vectors 2013;6:196.  Back to cited text no. 11
    
12.
Brahmachari UN. A new form of cutaneous leishmaniasis, dermal leishmanoid. Ind Med Gaz 1922;57:125-7.  Back to cited text no. 12
    
13.
Zijlstra EE, el-Hassan AM. Leishmaniasis in Sudan. Post kala-azar dermal leishmaniasis. Trans R Soc Trop Med Hyg 2001;95 (Suppl 1):S59-76.  Back to cited text no. 13
    
14.
The Post kala-azar dermal leishmaniasis (PKDL) Atlas-A manual for health workers. 2012. Available from: https://www.who.int/neglected_diseases/resources/9789241504102/en/. [Last accessed on 2020 May 20].  Back to cited text no. 14
    
15.
Zijlstra EE, Khalil EA, Kager PA, El-Hassan AM. Post kala-azar dermal leishmaniasis in the Sudan: Clinical presentation and differential diagnosis. Br J Dermatol 2000;143:136-43.  Back to cited text no. 15
    
16.
London Declaration on Neglected Tropical Diseases 2012. Available from: https://unitingtocombatntds.org/london-declaration-neglected-tropical-diseases/. [Last accessed on 2020 May 20].  Back to cited text no. 16
    
17.
Process of validation of elimination of kala-azar as a public health problem in South-East Asia, WHO, 2016. Available from: https://www.who.int/leishmaniasis/resources/Validation_of_elimination_of_VL_as_a_public_health_prob_SEARO/en/. [Last accessed on 2020 May 20].  Back to cited text no. 17
    
18.
Status of kala-azar in India. National Vector Borne Disease Control Programme (NVBDCP). Available from: https://nvbdcp.gov.in/index4.php?lang=1&level=0&linkid=467&lid=3750. [Last accessed on 2020 May 20].  Back to cited text no. 18
    
19.
Status of Kala-azar elimination programme (KAEP). Data obtained from Ministry of Health and family welfare. Government of India; 2018.  Back to cited text no. 19
    
20.
Ramesh V. On the differences between Post kala azar dermal leishmaniasis and leprosy. Trop Doct 1994;24:120-1.  Back to cited text no. 20
    
21.
Gama ME, Costa JM, Gomes CM, Corbett CE. Post-kala-azar dermal leishmaniasis. Int J Dermatol 1995;34:85-91.  Back to cited text no. 21
    
22.
Adams ER, Versteeg I, Leeflang MMG. Systematic review into diagnostics for Post kala-azar dermal leishmaniasis (PKDL). J Trop Med 2013;2013. doi: 10.1155/2013/150746.  Back to cited text no. 22
    
23.
Singh RP, Picado A, Alam S, Hasker E, Singh SP, Ostyn B, et al . Post-kala-azar dermal leishmaniasis in visceral leishmaniasis-endemic communities in Bihar, India. Trop Med Int Health 2012;17:1345-8.  Back to cited text no. 23
    
24.
Medley GF, Hollingsworth TD, Olliaro PL, Adams ER. Health-seeking behaviour, diagnostics and transmission dynamics in the control of visceral leishmaniasis in the Indian subcontinent. Nature 2015;528:S102-8.  Back to cited text no. 24
    
25.
Salotra P, Singh R. Challenges in the diagnosis of post kala-azar dermal leishmaniasis. Indian J Med Res 2006;123:295-310.  Back to cited text no. 25
    
26.
Sivakumar R, Dey A, Sharma P, Singh S. Expression and characterization of a recombinant kinesin antigen from an old Indian strain (DD8) of Leishmania donovani and comparing it with a commercially available antigen from a newly isolated (KE16) strain of L. donovani. Infect Genet Evol 2008;8:313-22.  Back to cited text no. 26
    
27.
Singh S, Kumari V, Singh N. Predicting kala-azar disease manifestations in asymptomatic patients with latent Leishmania donovani infection by detection of antibody against recombinant K39 antigen. Clin Diagn Lab Immunol 2002;9:568-72.  Back to cited text no. 27
    
28.
Maalej IA, Chenik M, Louzir H, Salah AB, Bahloul C, Amri F, et al . Comparative evaluation of ELISAs based on ten recombinant or purified Leishmania antigens for the serodiagnosis of Mediterranean visceral leishmaniasis. Am J Trop Med Hyg 2003;68:312-20.  Back to cited text no. 28
    
29.
Badaró R, Benson D, Eulálio MC, Freire M, Cunha S, Netto EM, et al . rK39: A cloned antigen of Leishmania chagasi that predicts active visceral leishmaniasis. J Infect Dis 1996;173:758-61.  Back to cited text no. 29
    
30.
Kumar D, Srividya G, Verma S, Singh R, Negi NS, Fragaki K, et al . Presence of anti-Lepp12 antibody: A marker for diagnostic and prognostic evaluation of visceral leishmaniasis. Trans R Soc Trop Med Hyg 2008;102:167-71.  Back to cited text no. 30
    
31.
Saha S, Mazumdar T, Anam K, Ravindran R, Bairagi B, Saha B, et al . Leishmania promastigote membrane antigen-based enzyme-linked immunosorbent assay and immunoblotting for differential diagnosis of Indian Post kala-azar dermal leishmaniasis. J Clin Microbiol 2005;43:1269-77.  Back to cited text no. 31
    
32.
Verma S, Bhandari V, Avishek K, Ramesh V, Salotra P. Reliable diagnosis of post kala-azar dermal leishmaniasis (PKDL) using slit aspirate specimen to avoid invasive sampling procedures. Trop Med Int Health 2013;18:268-75.  Back to cited text no. 32
    
33.
Ganguly S, Saha P, Chatterjee M, Roy S, Ghosh TK, Guha SK, et al . PKDL—A silent parasite pool for transmission of leishmaniasis in kala-azar endemic areas of Malda District, West Bengal, India. PLoS Negl Trop Dis 2015;9:e0004138.  Back to cited text no. 33
    
34.
Bhargava A, Ramesh V, Verma S, Salotra P, Bala M. Revisiting the role of the slit-skin smear in the diagnosis of Indian post kala-azar dermal leishmaniasis. Indian J Dermatol Venereol Leprol 2018;84:690-5.  Back to cited text no. 34
[PUBMED]  [Full text]  
35.
Ejazi SA, Bhattacharya P, Bakhteyar MAK, Mumtaz AA, Pandey K, Das VNR, et al . Noninvasive diagnosis of visceral leishmaniasis: Development and evaluation of two urine-based immunoassays for detection of Leishmania donovani infection in India. PLoS Negl Trop Dis 2016;10:e0005035.  Back to cited text no. 35
    
36.
Ejazi SA, Bhattacharyya A, Choudhury ST, Ghosh S, Sabur A, Pandey K, et al . Immunoproteomic identification and characterization of Leishmania membrane proteins as non-invasive diagnostic candidates for clinical visceral leishmaniasis. Sci Rep 2018;8:12110.  Back to cited text no. 36
    
37.
Osman OF, Oskam L, Kroon NC, Schoone GJ, Khalil ET, El-Hassan AM, et al . Use of PCR for diagnosis of Post kala-azar dermal leishmaniasis. J Clin Microbiol 1998; 36:1621-24.  Back to cited text no. 37
    
38.
Osman OF, Kager PA, Zijlstra EE, El-Hassan AM, Oskam L. Use of PCR on lymph-node samples as test of cure of visceral leishmaniasis treatment. Ann Trop Med Parasitol 1997;91:845-50.  Back to cited text no. 38
    
39.
Salotra P, Sreenivas G, Beena KR, Mukherjee A, Ramesh V. Parasite detection in patients with post kala-azar dermal leishmaniasis in India: A comparison between molecular and immunological methods. J Clin Pathol 2003;56:840-3.  Back to cited text no. 39
    
40.
Tavares CAP, Fernandes AP, Melo MN. Molecular diagnosis of leishmaniasis. Expert Rev Mol Diagn 2003;3:657-67.  Back to cited text no. 40
    
41.
Sreenivas G, Ansari NA, Kataria J, Salotra P. Nested PCR Assay for detection of Leishmania donovani in slit aspirates from post kala-azar dermal leishmaniasis lesions. J Clin Microbiol 2004;42:1777-8.  Back to cited text no. 41
    
42.
Mondal D, Nasrin KN, Huda MM, Kabir M, Hossain MS, Kroeger A, et al . Enhanced case detection and improved diagnosis of PKDL in a kala-azar endemic area of Bangladesh. PLoS Negl Trop Dis 2010;4:e832.  Back to cited text no. 42
    
43.
Singh S, Sharma U, Mishra J. Post kala-azar dermal leishmaniasis: Recent developments. Int J Dermatol 2011;50:1099-108.  Back to cited text no. 43
    
44.
Hassan MQ, Ghosh A, Ghosh SS, Gupta M, Basu D, Mallik KK, et al . Enzymatic amplification of mini-exon-derived RNA gene spacers of Leishmania donovani : Primers and probes for DNA diagnosis. Parasitology 1993;107:509-17.  Back to cited text no. 44
    
45.
Salotra P, Sreenivas G, Pogue GP, Lee N, Nakhasi HL, Ramesh V, et al. Development of a species-specific PCR assay for detection of Leishmania donovani in clinical samples from patients with kala-azar and Post kala-azar dermal leishmaniasis. J Clin Microbiol 2001;39:849-54.  Back to cited text no. 45
    
46.
Arora SK, Gupta S, Bhardwaj S, Sachdeva N, Sharma NL. An epitope-specific PCR test for diagnosis of Leishmania donovani infections. Trans R Soc Trop Med Hyg 2008;102:41-5.  Back to cited text no. 46
    
47.
Srivastava P, Mehrotra S, Tiwary P, Chakravarty J, Sundar S. Diagnosis of Indian visceral leishmaniasis by nucleic acid detection using PCR. PloS One 2011;6:e19304.  Back to cited text no. 47
    
48.
Nasreen SA, Hossain MA, Paul SK, Mahmud MC, Ahmed S, Ghosh S, et al . PCR-based detection of Leishmania DNA in skin samples of post kala-azar dermal leishmaniasis patients from an endemic area of Bangladesh. Jpn J Infect Dis 2012;65:315-7.  Back to cited text no. 48
    
49.
Verma N, Singh D, Pandey K, Das VN, Lal CS, Verma RB, et al . Comparative evaluation of PCR and imprint smear microscopy analyses of skin biopsy specimens in diagnosis of macular, papular, and mixed papulo-nodular lesions of post kala-azar dermal leishmaniasis. J Clin Microbiol 2013;51:4217-9.  Back to cited text no. 49
    
50.
Galluzzi L, Ceccarelli M, Diotallevi A, Menotta M, Magnani M, Magnani M. Real-time PCR applications for diagnosis of leishmaniasis. Parasit Vectors 2018;11:273.  Back to cited text no. 50
    
51.
Mary C, Faraut F, Lascombe L, Dumon H. Quantification of Leishmania infantum DNA by a real time PCR assay with high sensitivity. J Clin Microbiol 2004;42: 5249-55.  Back to cited text no. 51
    
52.
Sudarshan M, Weirather JL, Wilson ME, Sundar S. Study of parasite kinetics with antileishmanial drugs using real-time quantitative PCR in Indian visceral leishmaniasis. J Antimicrob Chemother 2011;66:1751-5.  Back to cited text no. 52
    
53.
Sudarshan M, Singh T, Chakravarty J, Sundar S. A correlative study of splenic parasite score and peripheral blood parasite load estimation by quantitative PCR in visceral leishmaniasis. J Clin Microbiol 2015;53:3905-7.  Back to cited text no. 53
    
54.
Verma S, Kumar R, Katara GK, Singh LC, Negi NS, Ramesh V, et al . Quantification of parasite load in clinical samples of leishmaniasis patients: IL-10 level correlates with parasite load in visceral leishmaniasis. PloS One 2010;5:e10107.  Back to cited text no. 54
    
55.
Schulz A, Mellenthin K, Schonian G, Fleischer B, Drosten C. Detection, differentiation, and quantitation of pathogenic Leishmania o rganisms by a fluorescence resonance energy transfer-based real-time PCR assay. J Clin Microbiol 2003;41:1529-35.  Back to cited text no. 55
    
56.
Mondal D, Ghosh P, Khan MA, Hossain F, Böhlken-Fascher S, Matlashewski G, et al . Mobile suitcase laboratory for rapid detection of Leishmania donovani using recombinase polymerase amplification assay. Parasit Vectors 2016;9:281.  Back to cited text no. 56
    
57.
Hossain F, Ghosh P, Khan MAA, Duthie MS, Vallur AC, Picone A, et al. Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of visceral leishmaniasis patients and the monitoring of their response to treatment. PloS One 2017;12:e0185606.  Back to cited text no. 57
    
58.
Moulik S, Chaudhuri SJ, Sardar B, Ghosh M, Saha B, Das NK, et al . Monitoring of parasite kinetics in Indian post-kala-azar dermal leishmaniasis. Clin Infect Dis 2017;66:404-10.  Back to cited text no. 58
    
59.
Ghosh P, Hasnain MG, Hossain F, Khan MAA, Chowdhury R, Faisal K, et al . Evaluation of real-time PCR for diagnosis of post kala-azar dermal leishmaniasis in endemic foci of Bangladesh. Open Forum Infect Dis 2018;5:ofy234.  Back to cited text no. 59
    
60.
Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 2002;16:223-9.  Back to cited text no. 60
    
61.
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al . Loop mediated isothermal amplification of DNA. Nucleic Acids Res 2000;28:E63.  Back to cited text no. 61
    
62.
Takagi H, Itoh M, Islam MZ, Razzaque A, Ekram ARSM, Hashighuchi Y, et al . Sensitive, specific, and rapid detection of Leishmania donovani DNA by loop-mediated isothermal amplification. Am J Trop Med Hyg 2009;81:578-82.  Back to cited text no. 62
    
63.
Mori Y, Kanda H, Notomi T. Loop-mediated isothermal amplification (LAMP): Recent progress in research and development. J Infect Chemother 2013;19:404-11.  Back to cited text no. 63
    
64.
Kaneko H, Kawana T, Fukushima E, Suzutani T. Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J Biochem Biophys methods 2007;70:499-501.  Back to cited text no. 64
    
65.
Verma S, Avishek K, Sharma V, Negi NS, Ramesh V, Salotra P. Application of loop-mediated isothermal amplification assay for the sensitive and rapid diagnosis of visceral leishmaniasis and post kala-azar dermal leishmaniasis. Diagn Microbiol Infect Dis 2013;75:390-5.  Back to cited text no. 65
    
66.
Verma S, Singh R, Sharma V, Bumb RA, Negi NS, Ramesh V, et al . Development of a rapid loop-mediated isothermal amplification assay for diagnosis and assessment of cure of Leishmania infection. BMC Infect Dis 2017;17:223.  Back to cited text no. 66
    
67.
Dixit KK, Verma S, Singh OP, Singh D, Singh AP, Gupta R, et al . Validation of SYBR green I based closed tube loop mediated isothermal amplification (LAMP) assay and simplified direct-blood-lysis (DBL)-LAMP assay for diagnosis of visceral leishmaniasis (VL). PLoS Negl Trop Dis 2018;12:e0006922.  Back to cited text no. 67
    
68.
Khan MGM, Bhaskar KRH, Salam MA, Akther T, Pluschke G, Mondal D. Diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for detection of Leishmania DNA in buffy coat from visceral leishmaniasis patients. Parasit Vectors 2012;5:280.  Back to cited text no. 68
    
69.
Ghasemian M, Gharavi MJ, Akhlaghi L, Mohebali M, Meamar AR, Aryan E, et al . Development and assessment of loop-mediated isothermal amplification (LAMP) assay for the diagnosis of human visceral leishmaniasis in Iran. Iran J Parasitol 2014;9:50.  Back to cited text no. 69
    
70.
Sriworarat C, Phumee A, Mungthin M, Leelayoova S, Siriyasatien P. Development of loop-mediated isothermal amplification (LAMP) for simple detection of Leishmania infection. Parasit Vectors 2015;8:591.  Back to cited text no. 70
    
71.
Mukhtar M, Ali SS, Boshara SA, Albertini A, Monnerat S, Bessell P, et al . Sensitive and less invasive confirmatory diagnosis of visceral leishmaniasis in Sudan using loop-mediated isothermal amplification (LAMP). PLoS Negl Trop Dis 2018;12:e0006264.  Back to cited text no. 71
    
72.
Adams ER, Schoone G, Versteeg I, Gomez MA, Diro E, Mori Y, et al . Development and evaluation of a novel loop-mediated isothermal amplification assay for the diagnosis of cutaneous and visceral leishmaniasis. J Clin Microbiol 2018;56:e00386-18.  Back to cited text no. 72
    
73.
Nzelu CO, Cáceres AG, Guerrero-Quincho S, Tineo-Villafuerte E, Rodriquez-Delfin L, Mimori T, et al . A rapid molecular diagnosis of cutaneous leishmaniasis by colorimetric malachite green-loop-mediated isothermal amplification (LAMP) combined with an FTA card as a direct sampling tool. Acta Trop 2016;153:116-9.  Back to cited text no. 73
    
74.
Imai K, Tarumoto N, Amo K, Takahashi M, Sakamoto N, Kosaka A, et al . Non-invasive diagnosis of cutaneous leishmaniasis by the direct boil loop-mediated isothermal amplification method and MinION™ nanopore sequencing. Parasitol Int 2018;67:34-7.  Back to cited text no. 74
    
75.
Mondal D, Hamano S, Hasnain MG, Satoskar A. Challenges for management of post kala-azar dermal leishmaniasis and future directions. Res Rep Trop Med 2014;5:105-11.  Back to cited text no. 75
    


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