Diagnostic Tools for Cutaneous Leishmaniasis: History
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Contributor:
Sachee Bhanu Piyasiri
,
Rajika Dewasurendra
,
Nilakshi Samaranayake
,
Nadira Karunaweera

Leishmaniasis, a neglected tropical disease, encompasses a spectrum of clinical conditions and poses a significant risk of infection to over one billion people worldwide. 

  • cutaneous leishmaniasis
  • Leishmania donovani
  • skin lesions
  • microscopy
  • diagnosis

1. Introduction

Leishmaniasis is a vector-borne disease caused by protozoan parasites of the genus Leishmania. It constitutes a spectrum of clinical presentations with three main clinical entities recognized; viz. cutaneous leishmaniasis (CL), muco-cutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL). CL is the most common, with an estimated annual incidence of 600,000 to 1 million new cases [1]. Typically, each Leishmania species is associated with a recognized clinical form in identified geographical setting(s). Old-World CL is caused by either Leishmania tropica or L. major while VL is caused by L. donovani (in the ISC, China, and Africa) and L. infantum (in the Mediterranean region) [2]. Post-kala-azar dermal leishmaniasis (PKDL), sometimes considered a fourth entity, is a sequel to VL caused by L. donovani and is reported in both the ISC and Africa [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17].
Leishmaniasis is targeted for elimination in the ISC, with a focus on VL due to L. donovani. However, the early diagnosis and management of other phenotypes of this Leishmania species will also be of importance in eliminating parasite reservoirs which could contribute to persistent disease transmission [3]. Diagnosing CL caused by L. donovani is challenging due to diverse clinical presentations mimicking other skin conditions, leading to misdiagnosis and delayed treatment. Conventional methods such as microscopy can yield false-negative results, while limited awareness among healthcare providers hampers recognition and reporting. Variable parasite loads in lesions hinder detection, and interpretation of results requires expertise to avoid false positives or negatives. Diagnostic tests involving parasite material or those relying on the immune responses of the host could potentially exhibit inadequate performance due to the incompatibility of the parasite strains used. Adopting advanced diagnostics often poses a challenge due to resource constraints. Delays in diagnosis may allow for disease progression as well as its further spread. Addressing these issues is vital for improving the diagnosis and management of this form of leishmaniasis.

2. The Diagnostic Tests Recommended by World Health Organization for Cutaneous Leishmaniasis

The World Health Organization (WHO) [18] provides guidelines for various approaches to diagnosing CL. Recommended diagnostic methods encompass parasitological techniques, including microscopy to visualize Leishmania parasites in stained smears, culture for parasite growth and identification, and PCR for detecting Leishmania DNA in lesion samples. Serological methods, such as the direct agglutination test (DAT) and ELISA, are utilized to detect antibodies against Leishmania antigens. Molecular methods, such as PCR, enable the identification of Leishmania DNA in skin or blood samples, while immunological approaches, exemplified by the immunofluorescent antibody test (IFAT), to identify Leishmania antibodies using fluorescence microscopy (Figure 1).
Diagnostics 13 02989 g001
Figure 1. Graphical representation of diagnostic methods of cutaneous leishmaniasis. PCR: polymerase chain reaction, RFLP: restriction fragment length polymorphism, LAMP: loop-mediated isothermal amplification. RPA: recombinase polymerase amplification assay. MLEE: multi-locus enzyme electrophoresis. MLMT: multi-locus microsatellite typing.
The choice of method may vary based on resource availability, prevalent Leishmania species, and healthcare expertise. Additionally, commercial test kits such as CL Detect rapid diagnosis kits are available for efficient diagnosis of CL. However, the gold standard for diagnosing CL remains parasitological diagnosis achieved by directly visualizing Leishmania parasites in clinical samples using microscopy, known for its high specificity [19].

3. Clinical Identification

In dermatological conditions, the appearance of the lesions and their conformity to recognized patterns is central in reaching a probable diagnosis, and CL is no exception. The type of lesions found in Sri Lanka have been widely described [20][21][22][23][24][25]. These are single, dry lesions on exposed parts of the body, which start as a papule and enlarge to a nodule with central ulceration over a period of 1–6 months. A silvery scab/crust covering the ulcer, which is easily removed and then re-forms, is also described. A number of accompanying features of these usually non-tender, non-itchy lesions are also frequently observed, such as hypo/hyper-pigmentation, induration, and erythema of the surrounding skin. The clinical presentation of CL due to L. donovani bears resemblance to cutaneous lesions caused by other Leishmania species responsible for typical CL, such as L. major, L. tropica, L. braziliensis, L. amazonensis, and L. mexicana. However, while there are noticeable similarities, there also exist discernible differences in the clinical characteristics of skin lesions [26][27][28][29][30][31][32][33][34][35][36][37].
Attempts have also been made to incorporate the clinical appearance and progression of the lesions into predictive scores aiding screening and diagnosis [38]. Overall, an acne-form, painless lesion progressing through the stages described above has been seen to have a comparatively high predictive value, while it is noteworthy that the sensitivity of most features in aiding a clinical diagnosis decreases as the lesion becomes more chronic. While self-resolving lesions have been reported [10], treatment of all patients with a parasitological diagnosis is recommended in Sri Lanka in view of the potential of the parasite species to cause systemic involvement through in vitro and ex vivo experiments [39]. Mucosal lesions, commonly involving the lips or the angle of the mouth, have also been reported in Sri Lanka as well as other foci reporting CL due to L. donovani [7][10][40]. The recorded number of VL due to L. donovani in Sri Lanka remains low [41] although there is a possibility that this condition may be under-diagnosed.

4. Direct Parasitological Methods for Diagnosis of CL

4.1. Direct Microscopy

The main methods include direct microscopic visualization of amastigotes in lesion aspirates (aspirating the lesion after injecting saline into it with a 23G needle), tissue scrapings (scraping the edge of the lesion using a sterile scalpel blade), impression smears using tissue biopsies, or isolation of parasites in culture in order to visualize the promastigote stage.

4.2. Histopathology

Histopathological diagnosis follows a standard procedure of obtaining a 2–3 mm punch biopsy from the active edge of the lesion and fixing the tissue in 10% formalin, followed by further processing where the tissue samples are dehydrated, cleared, embedded in paraffin (FFPE), cut into 4–5 μm thick sections and stained with hematoxylin and eosin. The amastigotes are visualized under a 100× oil immersion objective and typically seen inside tissue macrophages (histiocytes), while some may be also seen extra-cellularly.

4.3. Identification of Promastigotes by In Vitro Isolation (Parasite Cultures)

In vitro isolation of Leishmania sp. enables the definitive diagnosis of CL, which is important in proper clinical management. Culturing of Leishmania parasites, i.e., promastigotes, in suitable culture media has proven to be an effective method of diagnosis, with high sensitivity and specificity [10][17]. Culturing of parasites is also essential for further studies, such as parasite genomic studies, immune response evaluation, and vaccine candidate investigations.

4.4. Supplementary Tests to Histopathology

The detection of immune markers expressed by Leishmania using immunohistochemistry has been evaluated for the diagnosis of CL. Positive CD1a staining of amastigotes of Old World leishmaniasis by L. major and L. tropica has been previously reported [42]. In 2023, Riyal et al., identified a similar staining pattern in L. donovani causing CL for the first time, with a sensitivity of 74% [43]. While fluorescence in situ hybridization (FISH) is not a common approach in diagnosing CL, applying Leishmania genus-specific FISH probes on FFPE tissue has been shown to improve sensitivity over conventional staining [44].

5. Molecular Techniques for Identifying Leishmania Parasites

5.1. Polymerase Chain Reaction (PCR)

Several PCR-based protocols have been developed locally, to detect the local L. donovani parasite. PCR does not require abundant test material (i.e., cultured parasites) and the clinical samples obtained from patients could be directly subjected to PCR. This can be considered as a significant advantage of PCR-based methods.
Leishmania kinetoplast DNA contains small circular molecules called kDNA mini-circles. These are ideal targets for sensitive Leishmania detection due to their high copy numbers and conserved sequences. Despite challenges posed by diverse mini-circle networks, strain typing using this genomic region is complicated. Previously, characterizing Leishmania mini-circulomes required isolating and cloning before sequencing. A study by Kocher et al., in 2018 demonstrated that high-throughput sequencing of individual mini-circle PCR products bypasses these labor-intensive laboratory steps [45].

5.2. PCR–RFLP

PCR–RFLP is a molecular technique that involves digesting PCR-amplified products with restriction enzymes, generating unique polymorphic fragments that serve as markers for identifying Leishmania species [46]. PCR–RFLP methods have been used successfully to identify the causative agent of leishmaniasis up to species level, specifically using internal transcribed spacers (ITSs). ITSs are non-coding regions of rRNA genes which are known to be highly conserved among the Leishmania sp. That includes L. donovani, L. infantum, L. major, L. tropica and L. aethiopica. Hence, PCR amplification of the ITS region followed by restriction digestion by Hae III can be considered as a suitable method for identification and differentiation between all medically important Leishmania parasite species [47][48][49].

5.3. LAMP

Loop-mediated isothermal amplification (LAMP) is a method that amplifies DNA rapidly under isothermal conditions with high specificity and efficiency [50].
The LAMP assay was tested as a potential diagnostic tool to diagnose CL patients in Sri Lanka [50]. This study reported 82.6% sensitivity and 100% specificity with positive and negative predictive values of 100% and 66%, respectively, compared to microscopy as the gold standard. This proved to be a more efficient and cost-effective method, with an assay procedure that can be completed in 1 h and 40 min, compared to nested PCR which takes approximately 3 h and 30 min [51].

5.4. Recombinase Polymerase Amplification Assay (RPA)

Gunaratna et al., in 2018 [52], introduced a recombinase polymerase amplification-assay-based mobile suitcase laboratory, as a point-of-care diagnostic method for CL. This was tested with different sample types (i.e., punch biopsy, slit-skin smear and fine-needle aspirate) for optimal performance. The method was performed by the rapid extraction of DNA using SpeedXtract (Qiagen, Hilden, Germany) from patient samples and subjecting it to RPA assay using RPA primers specific to the kinetoplast mini-circle DNA of L. donovani.

5.5. Multi-Locus Enzyme Electrophoresis (MLEE)

The differences in the migration patterns of enzymes on a gel reflect the differences in single nucleotide polymorphisms (SNPs) in the genes encoding these enzymes and can be detected via gel electrophoresis. This is the basis of MLEE to identify SNPs in the coding genes [53]. Isoenzyme typing using MLEE was performed to identify the Leishmania species from an autochthonous VL patient in a study by Ranasinghe et al., 2012 [15].

5.6. Multi-Locus Microsatellite Typing (MLMT)

Isoenzyme typing of 15 different enzymes in two reference L. donovani strains (i.e., L. donovani MON-2 and MON-37) together with the suspected VL sample was performed using a method described by Alam in 2009 [54]. MLMT was employed to conduct a comparative analysis of L. donovani strains that fall under the MON-37 zymodeme. This comparison involved strains originating from Cyprus and Israel, as well as strains from the Indian subcontinent, the Middle East, China, East Africa, and strains belonging to other zymodemes [54]. The genetic analysis of the MON-37 strains revealed that those from Kenya, Sri Lanka, and India were more genetically similar to strains of other zymodemes from their respective regions than to MON-37 strains from different geographical areas. In contrast, MON-37 strains from Cyprus and Israel were distinct not only from each other but also from all the other MON-37 strains studied. This suggests that the Cyprus and Israel strains are likely indigenous to their respective regions [54].

6. Immunological Techniques

6.1. Enzyme-Linked Immunosorbent Assay (ELISA) Based Diagnostics

Highly sensitive in-house ELISA tests have been developed for the detection of CL, leading to improved sensitivity and specificity. Hartzell et al., in 2008 [55], conducted a study on soldiers with CL using a rK39 dipstick and the study revealed that some patients without clinical evidence of VL had a reactive rK39 assay result. This finding suggests a potential association between CL and the positivity of rK39 assay. On the other hand, a study by Svobodova et al., in 2009 [56], revealed all subjects testing negative in the rK39 assay for the transmission of an atypical form of CL caused by L. infantum in South Anatolia.

6.2. Immunochromatographic Strip Test (ICT)

rK39 antigen-based immunochromatographic strips, widely used for detecting VL infections, have been tested for their applicability in detecting CL infections. A study in Brazil using the commercial rK39 strip test (Kalazar DetectTM by In Bios International, Inc., Seattle, WA, USA) confirmed the absence of serological cross-reactions between individuals with CL and those with VL caused by L. infantum [57]. However, the same strip test was not found to be sensitive in detecting CL cases in Sri Lanka [39].
The peroxidoxin antigen has been recently used in ELISA-based diagnostic assays, including the CL Detect™ rapid test by Inbios International, USA. The CL Detect™ rapid test showed low sensitivity (31.3%) in its detection of L. donovani when tested on skin slits and dental broach samples in a study conducted in Ethiopia [58].

6.3. Direct Agglutination Test (DAT)

The direct agglutination test (DAT) has been widely used for more than 25 years as a diagnostic tool for VL and to some extent for the typical form of CL caused by L. major, L. tropica, L. mexicana, L. braziliensis and L. amazonensis. It has been found to have high clinical accuracy, with the accuracy of diagnosis depending on the titer of antibodies in the serum [59]. The presence of anti-Leishmania antibodies in the tested serum leads to the formation of a pale blue film over the well, indicating a positive result [60]. DAT can also be a useful addition to the diagnosis of CL due to L. donovani from serum samples [61].

7. Challenges in Diagnosing Atypical Cutaneous Leishmaniasis Caused by Leishmania donovani

Distinguishing CL from other skin conditions poses a significant diagnostic challenge [25]. The clinical spectrum of atypical presentations often mirrors various dermatological disorders, making accurate differentiation complex. Lesions may resemble eczema, psoriasis, fungal infections, or bacterial cellulitis, among others. This overlapping clinical manifestation can lead to misdiagnosis and subsequent delays in appropriate treatment [62]. Thus, a comprehensive understanding of the distinct features and a thorough evaluation of the clinical, microscopic, molecular, and immunological aspects of CL are essential for achieving precise diagnoses and facilitating effective management strategies. To circumvent this issue, healthcare providers should prioritize a comprehensive approach that combines clinical assessment, appropriate laboratory testing, and consideration of the patient’s travel history and the endemicity of the disease in the region.

8. Innovative Approaches for Diagnosis of Cutaneous Leishmaniasis

Saavendra et al.’s 2020 [63] study in Peru revealed that high-frequency ultrasound offered a promising approach for the non-invasive visualization of Leishmania (Viannia) braziliensis-induced CL. Their findings demonstrated a strong correlation between ultrasound findings and histopathological CL characteristics, suggesting that high-frequency ultrasound could be a reliable diagnostic tool for CL in resource-limited settings.

Furthermore, an artificial intelligence (AI)-based algorithm is currently utilized for the automated detection and diagnosis of leishmaniasis. In 2022, Zare et al. [64] devised an algorithm for Leishmania parasite detection using integral image representation, facilitating faster processing. The study achieved a recall rate of 65% and a precision rate of 50% for detecting leishmania-infected macrophages. This tool’s versatility extends to identifying unusual patterns of atypical CL skin lesions. 

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems are advanced tools for nucleic acid detection, offering high specificity, sensitivity, and speed. This technology is considered an ideal point-of-care test and it is versatile for various applications in the detection of CL in some geographical regions.

Buffi et al.’s 2023 study [65] presents a groundbreaking approach to improving leishmaniasis diagnostics with profound implications. Their utilization of high-resolution melting (HRM) analysis to pinpoint informative polymorphisms in single-copy genes encoding metabolic enzymes represents a significant leap forward, offering highly accurate and species-specific insights into Leishmania parasites, especially L. infantum. This precision is crucial for tailoring treatment strategies. Furthermore, the development of rapid genotyping assays based on HRM simplifies the genotyping process, replacing labor-intensive and specialized methods like multi-locus enzyme electrophoresis (MLEE) and multi-locus microsatellite typing (MLMT). 

9. Conclusions

Diagnostic options for atypical L. donovani-induced CL closely resemble those available for conventional CL. Direct microscopy of lesion material is the most cost-effective option which, along with the minimally invasive nature of sampling involved, makes it the method of choice to be implemented in resource-poor settings. Molecular methods (i.e., MLEE, RPA, NGS, LAMP), on the other hand, offer heightened sensitivity, crucial for detecting low parasite numbers in chronic or partially treated lesions and will help to correctly identify the Leishmania species. Point-of-care tests are the need of the day and the identification of candidate antigens with high immunogenicity combined with high sensitivity and specificity to be incorporated in rapid assays should be a priority. Moreover, advancing these assays to differentiate active disease from exposure for disease surveillance in endemic regions should also be explored. It is important to acknowledge the challenges associated with detecting atypical CL due to the differences in local L. donovani parasites.

This entry is adapted from the peer-reviewed paper 10.3390/diagnostics13182989

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