Application of CL Detect™ rapid test for diagnosis and liposomal amphotericin B for treatment of cutaneous leishmaniasis: A retrospective analysis from a tertiary care centre in a non-endemic area in India

Plain Language Summary

Cutaneous leishmaniasis is an infectious disease spread by the bite of infected sandflies. Affected individuals develop skin ulcers and can suffer from severe disfigurement. In recent years, the number of CL cases in non-endemic areas has increased due to increased migration and urbanisation. This article describes CL patients from different parts of India seen at a tertiary hospital in Delhi . This study evaluated the utility of the CL Detect Rapid test as a diagnostic tool and the efficacy of Liposomal Amphotericin B (LAmB) in treating CL patients to cure them completely. The CL detection test correctly identified nearly 80% of confirmed cases of CL. The infective species in these cases was Leishmania tropica. All the patients treated with LAmB responded to treatment, and 9/10 patients demonstrated complete regression of lesions, while one was lost to follow-up. The results of this study indicate that the CL Detect Rapid test is a reliable tool for diagnosing CL. Furthermore, LAmB is an effective treatment for CL, with a high success rate in curing the disease.

Introduction

Cutaneous leishmaniasis (CL), a vector-borne parasitic disease caused by Leishmania, affects the global population with 0.6–1.0 million cases annually.¹ In India, regions in and around the Thar desert in Rajasthan are considered endemic for CL. Additionally, cases from other areas, like Himachal Pradesh and Jammu & Kashmir, have been reported over the past two decades.^2–4

In the old world, the causative species are L. tropica, L. major and L. aethiopica.^1,5 Reports of atypical CL are well documented from Sri Lanka, where the infective species was L. donovani.⁶ Similarly, atypical CL due to L. donovani has been reported from Himachal Pradesh, India.²

Demonstration of Leishman-Donovan bodies (LDB), a standard test for diagnosis of CL in slit-skin smear from the lesion, has low sensitivity (40–60%) and needs expertise in interpretation.⁷ Immunological methods are not dependable for diagnosing CL.⁸ CL Detect™ Rapid Test is qualitative, in vitro immunochromatographic assay that detects peroxidoxin antigen of Leishmania species that has shown considerable sensitivity in detecting CL.^7,9,10 Nucleic acid amplification-based methods like PCR, real-time PCR and LAMP have been used to diagnose CL.^10,11 Molecular assays targeting parasite-specific Internal transcribed spacer (ITS) and heat shock protein (hsp) 70 are used for determining the parasite species.^10,12,13

Although CL self-heals, specific treatment can accelerate parasite clearance. Pentavalent antimony is the treatment of choice.¹⁴ Miltefosine is an oral drug with good efficacy, but a complete cure in old world CL is not common.¹⁵ Amphotericin B (AmB) is reserved for cases of antimony failures. Resistance to antimonials and side effects observed have supported the use of liposomal AmB (LAmB).^16,17 Other curative options, including local destructive measures like cryotherapy and heat therapy, require special equipment.^18,19 We have shown the utility of the CL Detect Rapid test, a less invasive procedure, as a promising diagnostic tool at the point of care. We also highlight the curative efficacy of LAmB.

Methods

Results

Demographic data and diagnostic tests

Of the 16 cases confirmed, 11 were males, and five were females. Fifteen cases hailed from Northern India (Rajasthan n = 6, Uttarakhand n = 4, Delhi n = 2, Haryana n = 1, Himachal Pradesh n = 1 and Uttar Pradesh n = 1) while one was from Nepal. The extent of CL observed was localised (n = 11) or disseminated (n = 5), as detailed in Table 1.

The onset of lesions at the reporting time varied from 2 months to 4 years. The lesions were erythematous nodules or raised plaques, with some showing thick central crusts. The most prevalent site was on the face in 14 cases. In the localised form, the eruptions were present on the face in all 11 patients; they were solitary in 7 and 2–3 in number in four. In the disseminated forms, either one or more of the extremities were affected; face was spared in 2 with many lesions in one patient being distributed on the right calf, and forearm and lower limb on the left side. In 13 of the 16 patients, the nodules had a sloping periphery and a central crater resembling a volcano. In some, the crust had fallen, revealing an erosion; in one case with disseminated lesions, the individual papules were flat or annular with central pits giving a honeycomb appearance. The other two had erythematous plaques with prominent irregular crust in one.

Histopathology showed normal to the atrophic epidermis with acanthosis in one case and parakeratosis in two cases. Dermis showed moderate to dense diffuse pan-dermal infiltrate of lymphocytes and histiocytes mixed with abundant plasma cells. Well- to ill-defined epithelioid cell granulomas were noted in 5 cases. Giemsa stain revealed LDB in 4/16 cases on histopathology and 7/16 slit-skin smears (sensitivity of 43.8%: 95% CI, 19.78–70.1%) [Figure 1].

Close

Figure 1a:

Dense lymphoplasmacytic infiltrate along with numerous LDB in the cytoplasm of histiocytes (H&E, 1000X).

Export to PPT

Close

Figure 1b:

Slit skin smear showing a solitary LD body (H&E, 400X).

Export to PPT

CL Detect Rapid Test performed in 16 confirmed CL cases were positive in 13 patients (81.3%; 95% CI, 54.4–96.0%). Although the assay is recommended for lesions not older than four months, it efficiently detected cases even when the lesions were older than four months. ITS-1 gene-based analysis identified L. tropica as the causative species in 12 samples.

Treatment

Intravenous infusion of LAmB was given in ten patients. After completion of three doses, the lesions showed 60% regression in 2–3 months, better appreciated in localised forms. Complete regression was seen in 6 months in three cases with localised CL [Figure 2]. The four disseminated and two localised forms took around 10 months for complete regression, while one case with localised CL, after taking all three doses, did not report for follow up. There was flattening of the nodules, a decrease in induration and re-epithelialisation of the ulcers. Complete re-epithelialisation was evident in the long-term follow-up [Figure 3]. Two patients were treated with SAG. One patient with a nodule on the upper lip responded to three intralesional injections of SAG. Another patient with disseminated CL, intolerant to LAmB, was given 8 ml (800 mg) SAG intravenously daily for 30 days.

Close

Figure 2a:

Localised cutaneous leishmaniasis – Pre-treatment.

Export to PPT

Close

Figure 2b:

Localised cutaneous leishmaniasis – One month post-treatment (with LAmB).

Export to PPT

Close

Figure 2c:

Localised cutaneous leishmaniasis – 10 months post-treatment complete re-epithelialisation.

Export to PPT

Close

Figure 3a:

Disseminated Cutaneous Leishmaniasis – Pre-treatment.

Export to PPT

Close

Figure 3b:

Disseminated Cutaneous Leishmaniasis – One month post-treatment (with LAmB).

Export to PPT

Close

Figure 3c:

Disseminated Cutaneous Leishmaniasis – 3 years post-treatment complete re-epithelialisation.

Export to PPT

Discussion

CL occurs mainly in resource-limited areas, and this study presents the cases that went undiagnosed during the initial visit of patients to doctors in endemic areas, mainly from parts of Rajasthan and Uttarakhand.

In expert hands, the positivity of LDB in slit-skin smears, better demonstrable in early lesions, is around 60%.^2,4,22 The plausible reason for the observed low positivity (44%) rate seen here is that many of the patients have taken antibiotics known to be leishmanicidal, e.g. rifampicin. Our observations are similar to the histomorphologic pattern in LDB-negative patients, where a high plasma cell density distributed diffusely and ill-formed granulomas should alert one to the possibility of CL.²³

The distinctive feature is the volcano sign seen in the lesions of most of our patients, in which the often elevated lesions show a central crater or crust mimicking a volcano.²⁴ It was also seen that the lesions evolved as groups of papules and characteristically revealed central pits resembling mini craters.²⁵ These features should raise the suspicion of CL in non-endemic areas since these are less common in other infective granulomas like tuberculosis and leprosy.^4,26

CL Detect Rapid test is an easily accessible tool, requiring little expertise in handling when performed with slit aspirate for sample collection. We observed the sensitivity of the CL Detect Rapid test to be 81.3%, higher than that reported in previous studies, possibly because slit aspirates from only confirmed cases of CL were subjected to the test.^7,10

The availability of antimonials is a problem, and alternative drugs like dapsone, rifampicin, and azoles are not consistent in their efficacy.²⁷ Further, some patients in our series had already taken these drugs. Hence LAmB, which had proven effective in both old and new-world CL, was evaluated and found to be highly effective.

Limitations

In this retrospective analysis, a small number of patients were analysed, and results on the performance of a CL Detect Rapid Test only on confirmed cases of CL and a short treatment with LAmB are presented.

Conclusion

This study demonstrated the application of the CL Detect Rapid test as a point-of-care test and also established the excellent performance of LAmB for treating CL.