Rapid Antigen Detection Test for Diagnosis of Post–kala-azar Dermal Leishmaniasis: Application of CL Detect Rapid Test for Active Case Detection in the Endemic Area

Abstract

The visceral leishmaniasis (VL) elimination program in the Indian subcontinent targeted the elimination of the disease. In 2023, Bangladesh was declared to have eliminated VL based on the number of reported cases being <1 per 10 000 population in each subdistrict for at least 3 consecutive years; however, the status of post–kala-azar dermal leishmaniasis (PKDL) was not available. A recent study from Bangladesh reported the overall PKDL burden as 1.3 per 10 000 population in the endemic villages [1]. According to the World Health Organization (WHO), cases of PKDL are a source of parasites that contribute to the transmission of VL. These need to be diagnosed and treated at an early stage. Patients with PKDL can also spread VL to nonendemic areas where the vector is present as they travel to such places for jobs [2].

PKDL is a dermatologic manifestation that develops in 5% to 10% of patients with cured VL from the Indian subcontinent and 50% to 60% from Sudan. PKDL presents as an asymptomatic facial erythematous and/or hypopigmented rash that may progress to papules and nodules or as polymorphic lesions that subsequently spread to the trunk, legs, and genital organs [3]. The WHO offers online courses and manuals to help frontline health workers identify PKDL in the field. However, diagnosing PKDL can be confounding due to other mimickers, underreporting, and low parasite load. The rK39 rapid diagnostic test (RDT) is commonly used to detect VL and PKDL. Yet, due to the presence of past antileishmanial antibodies resulting from previous VL episodes, the rK39 RDT cannot be used decisively as a diagnosis for PKDL [4]. Confirmatory tests for PKDL include histopathology and molecular testing, which demonstrate the presence of parasites in clinical samples. A highly sensitive, specific, and rapid test is undoubtedly needed to confirm active Leishmania infection in resource-constrained regions for diagnosis of cases of PKDL [2, 4].

The CL Detect Rapid Test for cutaneous leishmaniasis (CL), a Food and Drug Administration–approved in vitro diagnostic test, is an immunochromatographic assay that detects Leishmania antigen peroxidoxin, a member of thiol-specific antioxidant family proteins of several Leishmania spp, with detection limits ranging from 312 parasites for L tropica to 5000 parasite equivalents for L peruviana per strip. For L donovani, the analytic detection limit is 626 parasite equivalents [5, 6]. It is ideal for rural areas and takes only 30 minutes to complete on-site. It has been evaluated in various CL endemic regions with varying results [7–10]. Here, we assessed the utility of the CL Detect Rapid Test for diagnosing PKDL using a minimally invasive slit-skin-smear (SSS) sample collection approach.

METHODS

Study Period and Patients

Patients who presented with dermatoses at ICMR–Rajendra Memorial Research Institute of Medical Sciences, Patna, and ESIC Hospital, Faridabad, between February 2021 and February 2023 were included in the study. This study analyzed 75 PKDL cases (49 with polymorphic and 26 with macular presentation) and 63 dermatoses often confused with PKDL (viz leprosy, cutaneous tuberculosis, pityriasis rosea, tinea corporis, vitiligo, and sporotrichosis). The clinical samples positive by histopathology and/or real-time–polymerase chain reaction (RT-PCR) were considered cases of PKDL, while samples negative in both were classified as non-PKDL cases. The demographic and clinical characteristics of confirmed PKDL cases are described in Supplementary Table 1 and non-PKDL cases in Supplementary Table 2.

CL Detect Rapid Test

The CL Detect Rapid Test was carried out with slight changes to the sample collection method mentioned in the instruction manual. In this case, the SSS sample was collected by a sterile blade instead of a dental broach (Supplementary Figure 1A). SSS (50 µL) was mixed with 3 drops of lysis buffer and incubated for 5 to 10 minutes; then, 40 µL of lysate was applied onto the reservoir pad. The test strip was placed into a reaction tube with 3 drops of Chase Buffer Type A solution, and results were read in 20 to 30 minutes (Supplementary Figure 1B). The test was positive if control and test lines appeared, negative if only the control line appeared, and invalid if no control line was visible. The limit of detection of CL via a rapid test with L donovani isolates was determined with the parasite lysate prepared from varying numbers of cells (viz 10⁵, 10⁴, 10³, 10², and 10 parasites).

Real-time PCR Assay

Real-time PCR based on SYBR Green I with kDNA-based genus-specific primers was performed for quantification of the parasite following the methods described earlier [11]. DNA extraction was carried out with the DNeasy Blood and Tissue Kit (Qiagen) following the manufacturer's recommendations. PCR amplification and detection were performed with a BioRad CFX96 detection system. The PCR reaction (10 µL) consisted of 1× SYBR Green I PCR Master Mix, 5 pmol each of forward and reverse primer, and 2 µL of isolated DNA. Cycling conditions included 40 cycles at 95 °C (15 seconds) and 60 °C (1 minute) after an initial denaturation step at 95 °C (5 minutes), followed by a melt curve generation step.

Ethics Approval

The study was approved by the ethics committee of ICMR–National Institute of Pathology (NIP-IEC/18.06.20/03); ICMR–Rajendra Memorial Research Institute of Medical Sciences, Patna (RMRI/EC/14/20); and ESIC Hospital, Faridabad (134X/11/13/2021-IEC/47).

RESULTS

Among the confirmed PKDL cases, 54 (72%) patients were male, and 21 (28%) were female. The history of kala-azar was reported by 58 (77.33%) patients. The symptoms of PKDL presentation appeared a few months to as long as 31 years following subsidence of the systemic illness. All 75 confirmed patients were positive for RT-PCR, 67 of 75 (89.3%) were positive for anti-rK39 antibodies, whereas 57 of 75 (76.0%) were positive by histopathology.

The CL Detect Rapid Test detected up to 1000 L donovani parasite equivalents (Figure 1A). The test was highly specific: negative in all 63 tested dermatoses often confused with PKDL and positive in macular and polymorphic variants of PKDL (Figure 1B). Of the 26 macular PKDL cases, 16 (61.5%) were positive by the CL Detect Rapid Test, whereas the test was positive in 39 (79.5%) of the 49 polymorphic cases; the overall positivity rate for detection of leishmanial antigen in PKDL cases was 55 of 75 (73.5%; Table 1).

Figure 1.

A, Limit of detection of CL Detect Rapid Test with Leishmania donovani isolates. Lanes 1 and 2: positive and negative controls provided in kit. Lanes 3–7: lysate (10⁵, 10⁴, 10³, 10², and 10 parasites). B, CL Detect Rapid Test application on clinical samples. Lane 1: CL slit aspirate lysate. Lanes 2 and 3: PKDL slit aspirate lysate from nodular and macular lesion. Lanes 4–7: slit aspirates from other dermatoses (leprosy, vitiligo, cutaneous tuberculosis, and fungal infection) among patient samples. C indicates control line; T indicates test line. C, Agreement of CL Detect test. Left: LD bodies load in skin smear. The y-axis presents gradation of average parasite density per OIF: 0, nil parasites/1000 OIF; 1, 1–10 parasites/1000 OIF; 2, 1–10 parasites/100 OIF; 3, 1–10 parasites/10 OIF; 4, 1–10 parasites/OIF; 5, 10–100 parasites/OIF; 6, 100 parasites/OIF. Center: The y-axis presents RT-PCR Ct values. Right: The y-axis presents onset of PKDL after apparent clearance of visceral infection. Data are presented as mean (SEM). *P < .05. ^**P < .01. CL, cutaneous leishmaniasis; Ct, cycle threshold; LD, Leishman-Donovan; OIF, oil immersion field; PKDL, post–kala-azar dermal leishmaniasis; RT-PCR, real-time–polymerase chain reaction; TB, tuberculosis.

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We observed a significant correlation between positive results from the CL Detect test and the presence of Leishman-Donovan (LD) bodies in a microscopic examination (P = .004). We also observed that samples with lower RT-PCR cycle threshold values (P = .01) were more likely to test positive with the CL Detect test as compared with samples that tested negative (Figure 1C). The CL Detect test was able to detect PKDL cases regardless of the duration of the history of PKDL in a patient, whether 2 or 480 months. We did not find a direct correlation between the presence of new lesions and the positive results of the CL Detect test, unlike that in the case of CL [6].

DISCUSSION

Dealing with PKDL presents significant challenges because of the complexities involved in diagnosis and treatment. It is crucial to promptly diagnose and treat PKDL cases to achieve sustained elimination of kala-azar, as they play a pivotal role in maintaining the parasite reservoir during interepidemic periods of VL. A significant proportion of PKDL cases are often misdiagnosed at primary health centers. Diagnosing PKDL remains a major challenge due to sparse parasite loads, especially in macular cases. Conventional parasite detection methods, such as stained smears, histopathology, and culture, have low sensitivity, and serologic tests can be inconclusive due to the persistence of antileishmanial antibodies. Nucleic acid–based tests such as PCR, real-time PCR, and isothermal assays have addressed the limitation of conventional assays but are not widely available in endemic regions. These methods can be employed to assess the efficacy of novel antileishmanial medications and gauge the outcomes of therapies. Treatment of PKDL faces challenges, such as long treatment schedules and the emergence of antimony resistance, leading to the use of miltefosine as a first-line treatment, which has seen increased relapse rates, side effects, and poor patient compliance [12].

In India, there are reports of sporadic cases of VL and PKDL from nonendemic areas as well, and diagnosis is quite challenging, as the gold standard, microscopy, requires highly trained personnel and molecular assays are expensive. Although the rK39 RDT test showed high positivity, it is not recommended for detecting cases of PKDL, as serologic tests such as rK39 RDTs can be positive due to past VL infection. Experts recommend using point-of-care tests that target Leishmania antigen for detecting dermal leishmaniasis [13]. The CL Detect Rapid Test, an antigen-based assay, had 73.3% sensitivity and 100% specificity in our study, similar to histopathology. The highly significant correlation between the positivity of the CL Detect test and the presence of LD bodies under microscopy suggests that the test can be used as a primary screening tool for PKDL at the field site. Further improvements in the test to achieve the required sensitivity of >90% would enhance its value as a point-of-care tool for the VL elimination program.

In this proof-of-concept study, we demonstrated the utility of the CL Detect test for diagnosing PKDL. However, the study has limitations in terms of a statistically valid sample size. A more comprehensive study with a larger sample size and a wider range of skin disease conditions relevant to the differential diagnosis of PKDL is required.

The current VL elimination framework proposes 5 main strategies for guiding VL elimination: early diagnosis and treatment; integrated vector management; effective surveillance; advocacy, social mobilization, and partnership building; and implementation and operational research [14]. The 2 key interventions—first, the early diagnosis made feasible by a highly sensitive and specific and quickly conducted rK39 test; second, the free availability of the single-dose liposomal amphotericin B and oral miltefosine to patients with VL—have tremendously contributed to the success of the VL elimination program. PKDL was not initially included as a direct target for elimination, resulting in its frequent neglect and oversight during active case searches. However, the latest WHO neglected tropical disease road map for eliminating VL includes a subtarget to detect PKDL cases—specifically, VL cases to be followed up for 3 years and ensure their complete treatment by 2030 [15]. A quick and easy antigen-based test for PKDL would be invaluable for monitoring endemic regions and identifying active Leishmania infection.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

Acknowledgments. CL Detect Rapid Test kits were provided by InBios International, Inc.

Author contributions. R. S., P. S., and V. R. contributed conceptualization. M. A., K. K.D., and T. G. performed experimental work. R. S. and V. N. R. D. supervised laboratory work and performed data curation and analysis. V. N. R. D., R. K. T., and V. R. coordinated the clinical samples. R. S. and M. A. wrote the original draft. All authors critically read, revised, and approved the final manuscript.

Disclaimer. The funder has no role in the study design or collection, analysis, and interpretation of data; the writing of this article; or the decision to submit it for publication.

Financial support. This work was supported by the Indian Council of Medical Research by grant 6/9-7(228)2020-ECDII.

References

Author notes