False Positivity of rK39 Test in Five Chronic Myeloid Leukemia Cases from Bihar, India: A Possible Challenge to Leishmaniasis Diagnosis.

A rapid and noninvasive rK39 rapid diagnostic test (RDT) is the best and most reliable tool for visceral leishmaniasis (VL) screening in the field. However, splenic and bone marrow aspiration remain two gold standard methods for microscopic identification of Leishmania donovani (LD) bodies and confirmatory diagnosis of VL. Five patients with signs and symptoms of fever, loss of appetite, loss of weight, hepatomegaly, and massive splenomegaly were found to be false positive with the rK39 RDT. These patients were suspected to have chronic myeloid leukemia (CML) because their blood pictures showed a total white blood cell count of > 100,000/mm3 and abnormal cells such as stab, segmented promyelocytes, myelocytes, metamyelocytes, and blast cells. Splenic aspirate and bone marrow were negative for Leishmania donovani bodies. The bone marrow showed myeloid series of cells, that is, myelocytes, metamyelocytes, stab and segmented cells, blast cells, and markedly increased myeloid:erythroid ratio. Later, the CML diagnosis was confirmed in all cases by breakpoint cluster region-tyrosine protein kinase (BCR-ABL) gene positive test results. In this study, the rK39 RDT's false positivity was observed in CML cases. It could have important implications for the differential diagnosis of VL with CML. The rK39 positive test result in CML cases was a serendipitous occurrence; this should be validated further to determine the utility of the rK39 test in the differential diagnosis of VL with CML.

Cathelicidin augments VDR-dependent anti-leishmanial immune response in Indian Post-Kala-Azar Dermal Leishmaniasis.

OBJECTIVES: Indian Post kala-azar dermal leishmaniasis (PKDL) is the cutaneous aftermath of visceral leishmaniasis (VL) caused by L. donovani. Vitamin D-regulated cationic antimicrobial peptide cathelicidin (hCAP-18/LL-37) has microbicidal and immunomodulatory role against cutaneous infections, but its role in PKDL remains elusive. METHODS: Skin snips and blood-derived monocytes of PKDL patients (n=46), before (BT) and after (AT) chemotherapy, were used for this study. Serum vitamin D3 level was evaluated by ELISA. Cathelicidin and vitamin D receptor (VDR) levels were analyzed by real-time PCR and flowcytometry in PKDL patients. The mechanistic effect of cathelicidin on macrophage differentiation and anti-leishmanial activity was assessed through RNA interference techniques followed by subsequent microscopic evaluation of in vitro parasite killing and Th1/Th2 counter-regulation by ELISA/RT-PCR. RESULTS: Low vitamin D3 levels were accompanied with decreased expression of cathelicidin and VDR in PKDL-BT patients. Results suggested positive induction of VDR-dependent cathelicidin in PKDL macrophages by Amphotericin B treatment, which could be due to indirect effect of drug-induced IL12 upregulation. 1,25-Vitamin D3 stimulation induced cathelicidin in PKDL-BT patients through involvement of TLR2/IL-1ß, but not TLR4. Cathelicidin also augmented the anti-leishmanial effect and macrophage activating potential of Amphotericin B, attributable to regulation of VDR-dependent enhancement of CD40, p-STAT-I and MHC-II expression leading to regulation of IL10/IL12 balance in PKDL-BT patient macrophages. CONCLUSIONS: This study indicates that cathelicidin augments anti-leishmanial macrophage activating property of Amphotericin B in a TLR2/VDR dependent mechanism, and advocate the development of novel adjunct treatment modality of cathelicidin with conventional Amphotericin B in PKDL patients.

Chronic Arsenic Exposure and Risk of Post Kala-azar Dermal Leishmaniasis Development in India: A Retrospective Cohort Study.

BACKGROUND: Visceral leishmaniasis (VL), with the squeal of Post-kala-azar dermal leishmaniasis (PKDL), is a global threat for health. Studies have shown sodium stibogluconate (SSG) resistance in VL patients with chronic arsenic exposure. Here, we assessed the association between arsenic exposure and risk of developing PKDL in treated VL patients. METHODS: In this retrospective study, PKDL patients (n = 139), earlier treated with SSG or any other drug during VL, were selected from the study cohort. Trained physicians, unaware of arsenic exposure, interviewed them and collected relevant data in a questionnaire format. All probable water sources were identified around the patient's house and water was collected for evaluation of arsenic concentration. A GIS-based village-level digital database of PKDL cases and arsenic concentration in groundwater was developed and individual point location of PKDL cases were overlaid on an integrated GIS map. We used multivariate logistic regression analysis to assess odds ratios (ORs) for association between arsenic exposure and PKDL development. RESULTS: Out of the 429 water samples tested, 403 had arsenic content of over 10 µg/L, with highest level of 432 µg/L among the seven study villages. Multivariate adjusted ORs for risk of PKDL development in comparison of arsenic concentrations of 10.1-200 µg/L and 200.1-432.0 µg/L were 1.85 (1.13-3.03) and 2.31 (1.39-3.8) respectively. Interestingly, similar results were found for daily dose of arsenic and total arsenic concentration in urine sample of the individual. The multivariate-adjusted OR for comparison of high baseline arsenic exposure to low baseline arsenic exposure of the individuals in the study cohort was 1.66 (95% CI 1.02-2.7; p = 0.04). CONCLUSION: Our findings indicate the need to consider environmental factors, like long time arsenic exposure, as an additional influence on treated VL patients towards risk of PKDL development in Bihar.

Specific noninvasive detection of Leishmania donovani in desquamated buccal cell swab samples from human visceral Leishmaniasis-HIV coinfected patients.

Diagnosis of human immunodeficiency virus (HIV) infection with visceral leishmaniasis (VL) coinfection is challenging. Specific diagnosis of VL in HIV-coinfected patients was evaluated by molecular methods in desquamated buccal swab samples, demonstrating 86.3% sensitivity and 98.3% specificity in controls. This test holds significant potential for development as a noninvasive diagnostic tool for VL in HIV-coinfected patients.

Combination of paromomycin and miltefosine promotes TLR4-dependent induction of antileishmanial immune response in vitro.

OBJECTIVES: To evaluate the in vitro activity of antileishmanial drugs, paromomycin and miltefosine, to generate Th-1-biased immunomodulation in hosts against intracellular Leishmania donovani. METHODS: In silico protein-ligand interaction and in vitro drug-cell interaction assays were performed. Interaction assays of TLR4-deficient HEK293 cells and HEK293 cells engineered to express either TLR4 or TLR2 with different concentrations of miltefosine and/or paromomycin sulphate were performed for 48 h. Differentially transfected human peripheral blood monocyte-derived macrophages (PBMFs) were treated with the drugs, and nuclear factor (NF)-κB promoter activity was measured using a κB-luciferase reporter construct. PBMFs were infected with L. donovani. Cultures were incubated with miltefosine or paromomycin sulphate over different concentrations, as mono-treatment or combined. The in vitro antileishmanial effect of the drugs on macrophage-bound L. donovani amastigotes was measured in terms of parasite killing and production of tumour necrosis factor-α (TNF-α) and nitric oxide. RESULTS: Computational studies reveal that paromomycin and miltefosine interact with TLR4. Both drugs, as monotherapy or in combination, induce release of TNF-α and nitric oxide in a TLR4-dependent manner. Interestingly, the TLR4-dependent action of the drugs leads to NF-κB promoter activation through MyD88. In vitro, both the drugs kill macrophage-bound L. donovani by inducing release of TNF-α and nitric oxide in a TLR4-dependent manner. CONCLUSIONS: The in vitro activity of paromomycin and miltefosine against host cells is TLR4 dependent. This has implications for: (i) evaluation of in vitro activity of combinational antileishmanial therapy; (ii) the evaluation of drug susceptibility of clinical isolates; and (iii) the standardization of in vitro antileishmanial assays for determining toxicity in hosts.