Identification of atypical dermal leishmaniasis resolved by restriction fragment length polymorphism.

This case report series alerts to the atypical manifestations of dermal leishmaniasis in an area endemic for post kala-azar dermal leishmaniasis, the sequel to visceral leishmaniasis. We have reported two cases with multiple skin lesions, wherein the rK39 strip test, polymerase chain reaction and parasite load confirmed the presence of Leishmania parasites. The causative parasite was identified as Leishmania major by restriction fragment length polymorphism of the ribosomal DNA Internal Transcribed Spacer-1, overruling the clinical suspicion of post kala-azar dermal leishmaniasis. The third case presented with fever and extensive hypopigmented patches in the upper extremities; parasites were identified in blood and skin by polymerase chain reaction and typed by restriction fragment length polymorphism as Leishmania donovani, establishing this as a case of visceral leishmaniasis concomitant with dermal leishmaniasis, secondary to dissemination of viscerotropic L. donovani. The present case series emphasizes the importance of molecular tools to identify the Leishmania species in order to ensure appropriate treatment.

Glycoproteins in circulating immune complexes are biomarkers of patients with Indian PKDL: A study from endemic districts of West Bengal, India.

BACKGROUND: Post Kala Azar Dermal Leishmaniasis (PKDL) occurs as dermal consequence of previous Visceral Leishmaniasis (VL) infection and serves as an important reservoir for transmission of VL. Diagnosis of PKDL is often challenging for its symptomatic resemblance to other co-endemic diseases like Leprosy or Vitiligo. Parasitological examination by slit-skin smear and culture are the standard methods but lack high sensitivity. Thus, for efficient control of VL, reliable diagnostic and prognostic assay of PKDL are required. OBJECTIVE: Previously, glycoproteins (9-OAcSA) have been reported as promising biomarkers of Indian VL patients. However, till date, the status of glycans in Indian PKDL patients remains unexplored. Accordingly, in this study, the glyco-profile of PKDL Circulating Immune Complexes (CICs) as compared to other cross diseases like Vitiligo and Leprosyhas been investigated. Further, a novel Glyco CIC assay has been developed for efficient Indian PKDL patient diagnosis. METHODS/PRINCIPAL FINDING: In the present study, 90 PKDL patients were enrolled from 3 VL endemic districts of West Bengal during 2015-16. Glycosylation profile of isolated CICs from sera of PKDL patients were initially analyzed through gradient SDS gel electrophoresis followed by PAS silver double staining, which revealed the presence of several glycan rich PKDL specific proteins of varying molecular weights. To further characterize the glyco-profile of acid dissociated affinity purified immuno-reactive antigens present in the CICs, glycosylation was demonstrated in these purified CIC antigens by DIG glycan differentiation kit with or without glycosidase as well as neuraminidase treatment. Diagnostic evaluation of the newly developed colorimetric Glyco CIC assay through Receiver Operating Characteristic (ROC) curve analysis revealed excellent (0.99) AUC value as compared to other conventional serodiagnostic assays like PEG CIC, Parasite ELISA (IgG and IgM). Additionally, longitudinal monitoring of 18 PKDL patients further revealed its good prognostic utility. CONCLUSION: These results highlight the glycosylation status of CICs among Indian PKDL patients present in all the studied endemic districts of West Bengal. These PKDL biomarkers were completely absent in cross diseases like Vitiligo and Leprosy. Further, the newly developed Glyco CIC assay had an improved sensitivity of 95.6%, specificity of 99.3%, NPV of 97.1% and PPV of 98.9%.

Monitoring of Parasite Kinetics in Indian Post-Kala-azar Dermal Leishmaniasis.

Background: The potential reservoirs of leishmaniasis in South Asia include relapsed cases of visceral leishmaniasis (VL), patients with post-kala-azar dermal leishmaniasis (PKDL), and an asymptomatically infected population. Therefore, assessment of cure in terms of parasite clearance, early detection of PKDL, and asymptomatic VL are pivotal for ensuring elimination. This study aimed to monitor the efficacy of miltefosine and liposomal amphotericin B (LAmB) in PKDL based on parasite load. Methods: Patients with PKDL were recruited from the dermatology outpatient departments or during active field surveys. Skin biopsies were collected at disease presentation, immediately at the end of treatment, and 6 months later. The presence of parasite DNA was assessed by internal transcribed spacer-1 polymerase chain reaction, and quantified by amplification of parasite kinetoplastid DNA. Results: At disease presentation (n = 184), the median parasite load was 5229 (interquartile range [IQR], 896-50898)/µg genomic DNA (gDNA). Miltefosine cleared the parasites to <10 in the macular (n = 17) and polymorphic (n = 21) variants, and remained so up to 6 months later (<10 parasites). LAmB reduced the parasite burden substantially in macular (n = 34; 2128 [IQR, 544-5763]/µg gDNA) and polymorphic PKDL (n = 36; 2541 [IQR, 650-9073]/µg gDNA). Importantly, in patients who returned 6 months later (n = 38), a resurgence of parasites was evident, as the parasites increased to 5665 (IQR, 1840-17067)/µg gDNA. Conclusions: This study established that quantifying parasite load is an effective approach for monitoring patients with PKDL, wherein miltefosine demonstrated near-total parasite clearance and resolution of symptoms. However, in cases treated with LAmB, the persistence of parasites suggested treatment inadequacy. This needs immediate redressal in view of the leishmaniasis elimination program targeted for 2020.