Development of a rapid loop-mediated isothermal amplification assay for diagnosis and assessment of cure of Leishmania infection.

BACKGROUND: Leishmaniasis is a spectrum of diseases with great relevance to public health. Conventional diagnostic methods are time consuming, needing trained personnel. A robust, rapid and cost effective diagnostic test is warranted for on-time diagnosis and field application. METHODS: We have developed a loop mediated isothermal amplification (LAMP) assay with primers (n = 6) based on Leishmania donovani kDNA for detection of Leishmania infection, using a closed tube to prevent cross-contamination. The assay was used to detect Leishmania infection in biological samples obtained from patients of visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL). RESULTS: The assay was positive for L. donovani, L. tropica and L. major parasites, with the highest sensitivity towards L. donovani (1 fg DNA). The high sensitivity of the assay for detection of L. donovani was reflected in its ability to detect parasite DNA within 30 min of amplification time with a threshold detection limit of ≥25 copies per reaction. The assay detected parasite in 64 of 66 VL blood samples (sensitivity, 96.9%; 95% CI: 89.6-99.2%), 15 of 15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI:79.6-100%), 65 of 67 PKDL tissue biopsy samples (sensitivity, 97%; 95% CI:89.7-99.2%). The assay was evaluated in a few cases of CL wherein it was found positive in 8 of 10 tissue biopsies (sensitivity, 80%; 95% CI: 49-94.3%). The assay was negative in all control blood (n = 76) and tissue biopsy (n = 24) samples (specificity, 100%; 95% CI: 96.3-100%). Further, the assay was evaluated for its utility in assessment of cure in treated VL and PKDL patients. The assay detected parasite DNA in 2 of 20VL blood samples and 2 of 21 PKDL tissue samples. Out of 4 cases that were positive for parasite DNA at post treatment stage, 2 patients (1VL and 1 PKDL) returned with relapse. CONCLUSIONS: The study demonstrated a Leishmania genus specific closed tube LAMP assay for reliable and rapid molecular diagnosis of VL and PKDL with potential for application in assessment of cure.

Increased parasite surface antigen-2 expression in clinical isolates of Leishmania donovani augments antimony resistance.

Resistance to sodium antimony gluconate (SAG) is a major cause of therapeutic failure in a large proportion of visceral leishmaniasis (VL) cases. Determinants of SAG resistance have been widely studied; however, the mechanism operating in clinical isolates is poorly understood. In the present study, expression of parasite surface antigen-2 (PSA-2) gene was studied in clinical isolates of Leishmania donovani comprising of antimony resistant (n=10) and sensitive (n=4) parasites. The expression of PSA-2 gene was found to be consistently high in SAG resistant clinical isolates (≥1.5-fold) at both transcript and protein level. Further, over-expression of PSA-2 in L. donovani isolates (LdPSA-2(++)) resulted in conversion of SAG sensitive phenotype to resistant. The LdPSA-2(++) parasites showed significantly decreased susceptibility towards SAG (>12-fold), amphotericin B (>4-fold) and miltefosine (>2.5-fold). Marked decrease in antimony accumulation and enhanced tolerance towards complement mediated lysis was evident in LdPSA-2(++) parasites. The study established the role of PSA-2 gene in SAG resistance and its potential as a biomarker to distinguish resistant and sensitive clinical isolates of L. donovani.

Biomarkers of antimony resistance: need for expression analysis of multiple genes to distinguish resistance phenotype in clinical isolates of Leishmania donovani.

Resistance to antimony is a major cause of failure to therapy in a large proportion of visceral leishmaniasis cases. Methods to distinguish resistant and sensitive parasite are urgently needed as the standard in vitro intracellular drug susceptibility assays are cumbersome and time consuming. Differential expression profiling studies have led to the identification of several antimony resistance-associated genes; however, their efficacy as a potential biomarker for monitoring antimony resistance remains imprecise. We analysed the expression of eight genes [antimony metabolism-associated genes - multidrug resistance protein A (MRPA), γ-glutamylcysteine synthetase (γ-GCS) and aquaporin-1 (AQP1) - and genes identified by proteome/transcriptome profiling­heat shock protein 83, mitogen-activated protein kinase 1 and histones H1, H2A and H4) in antimony-resistant (n=10) and antimony-sensitive (n=4) clinical isolates of Leishmania donovani by quantitative real-time PCR, in comparison with a lab-generated resistant and a standard sensitive isolate. We observed a significant differential expression of MRPA, histone H1 (p<0.01), γ-GCS, HSP83 (p<0.005) and histone H2A and H4 (p<0.0001) in a group of sodium antimony gluconate-resistant isolates compared to sensitive isolates. Preferential AQP1 expression was observed in all the sensitive isolates (p<0.0001). Overall, expression profile in field isolates for all the genes studied showed altered expression in majority of isolates, while in some, the expression was static. All the isolates showed a mosaic of expression pattern of the genes analysed indicating constellation of genes contributes towards the drug susceptibility of parasite. As none of the genes exhibit an absolute correlation with phenotype, targeted expression analysis of a set of genes should be considered as biomarker for distinguishing the antimony-resistant and antimony-sensitive parasite.

Real-Time Fluorimetry Loop-Mediated Isothermal Amplification for Diagnosis of Leishmaniasis and as a Tool for Assessment of Cure for Post-Kala-Azar Dermal Leishmaniasis.

Despite the dwindling number of visceral leishmaniasis (VL) cases in India, there is an urgent need for early and unequivocal diagnostics for controlling and preventing the reemergence of VL. Post-kala-azar dermal leishmaniasis (PKDL), a dermal sequela of VL, serves as a reservoir of the parasite. Diagnosis of PKDL, especially the macular variant, is challenging and poses impediment toward attainment of VL elimination. In this study, a real-time fluorimetry loop-mediated isothermal amplification (RealAmp) assay has been established for the detection of different clinical manifestations of leishmaniasis. The study included 150 leishmaniasis patients (25 VL, 25 cutaneous leishmaniasis [CL], and 100-PKDL) along with 120 controls. The assay demonstrated sensitivity of 100% (95% CI: 86.68-100) for diagnosis of VL and PKDL (95% CI: 79.61-100) and 96% (95% CI: 86.68-100) for CL with 100% specificity. Moreover, considering the cardinal role of PKDL, diagnosis using minimally invasive slit aspirate was explored, which demonstrated remarkable sensitivity of 96% (95% CI: 87.64-98.47). As a test of cure for PKDL, RealAmp successfully detected parasite in two of posttreatment cases who later reported relapse on follow-up. Also, direct sample lysis using slit aspirate was attempted in a small group that yielded sensitivity of 89% (95% CI: 67.20-96.90). RealAmp depicted excellent diagnostic accuracy in the diagnosis of leishmaniasis in concordance with the established SYBR Green I-based (Molecular Probes, Eugene, OR) visual loop-mediated isothermal amplification (LAMP) and the reference comparator real-time PCR. The study endorsed the employment of LAMP either as visual-LAMP or RealAmp for an accurate and expeditious diagnosis of PKDL and as a tool for assessment of cure.

Presence of anti-Lepp12 antibody: a marker for diagnostic and prognostic evaluation of visceral leishmaniasis.

The diagnostic potential of recombinant Lepp12 (rLepp12) antigen cloned from Leishmania infantum was assessed in L. donovani infections by Western blotting. Ninety-two serum samples, including 30 patients with active kala-azar (KA), 17 post-treated KA patients (KA-PT), 20 post-kala-azar dermal leishmaniasis (PKDL) patients and 25 controls, were analysed for rLepp12, rK39 and DAT positivity. All KA samples taken at pre-treatment stage were positive for Lepp12 antibodies. Seventeen of these were evaluated post treatment (KA-PT), 10 of which were found to be negative. Nine of these 10 negative cases corresponded to clinically cured patients with regressed spleen. Seven post-treatment cases were rLepp12-positive; all of them corresponded to patients who were considered clinically cured but continued to have an enlarged spleen (> or =5 cm). The majority of PKDL patients (18/20) were found to be seronegative by immunoblot test using rLepp12 antigen. The rLepp12-based Western blot diagnosed 100% of patients with visceral disease, whilst none of the control cases were found to be reactive to rLepp12. rLepp12 protein provides a useful reagent for highly sensitive and specific diagnosis of KA. Additionally, rLepp12 appears to have potential as a prognostic marker for the infection.

Validation of SYBR green I based closed tube loop mediated isothermal amplification (LAMP) assay and simplified direct-blood-lysis (DBL)-LAMP assay for diagnosis of visceral leishmaniasis (VL).

BACKGROUND: The World Health Organization has targeted elimination of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) by 2020. Despite distinctive decline seen in the number of VL cases in ISC, there is still a quest for development of a diagnostic test which has the utility for detection of active infection and relapse cases and as a test of cure. The present study validated the sensitivity and specificity of SYBR Green I based closed tube LAMP assay reported by us for diagnosis of VL. METHODOLOGY: The validation study was carried out at two endemic sites in India, located at Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna and Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi. Standard operating protocols were provided at the two sites for applying LAMP assay on confirmed VL cases. The diagnostic accuracy of LAMP assay was evaluated by Receiver operator curve (ROC) analysis. Furthermore, a simplified LAMP assay based on direct blood lysis, DBL-LAMP, was developed and verified for its diagnostic accuracy. PRINCIPAL FINDINGS: A total of 267 eligible participants were included in the study which comprised of 179 VL cases and 88 controls. Sensitivity and specificity of the LAMP assay were 98.32% (95% C.I- 95.2-99.7%) and 96.59% (95% C.I.-90.4-99.3%), respectively. ROC curve analysis depicted no significant difference between area under curve (AUCROC) for LAMP assay and rK39 RDT, indicative of LAMP as an excellent diagnostic test. DBL-LAMP assay, performed on 67 VL and 100 control samples, yielded a sensitivity of 93.05% (95% C.I- 84.75-97%) and specificity of 100% (95% C.I.- 96.30-100%). CONCLUSIONS/SIGNIFICANCE: The validated closed tube LAMP for diagnosis of VL will provide impetus to the ongoing VL elimination programme in ISC. The assay based on direct blood lysis promotes its scope for application in field settings by further reducing time and cost.

Application of loop-mediated isothermal amplification assay for the sensitive and rapid diagnosis of visceral leishmaniasis and post-kala-azar dermal leishmaniasis.

Loop-mediated isothermal amplification (LAMP) is at the forefront in the search for innovative diagnostics for rapid and specific amplification of target DNA under isothermal conditions. We have applied LAMP assay using SYBR Green for clear-cut naked eye detection of Leishmania (Leishmania) donovani in 200 clinical samples of visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). The assay was positive in 53/55 VL blood samples (sensitivity, 96.4%; 95% confidence interval [CI], 87.7-99%), 15/15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI, 79.6-100%), 60/62 PKDL tissue biopsy samples (sensitivity, 96.8%; 95% CI, 88.9-99.1%), and 1/68 control samples (specificity, 98.5%; 95% CI, 92.1-99.7%). The assay was specific for L. (L.) donovani, the causative species for VL and negative for L. (L.) infantum, L. (L.) tropica, and L. (L.) major. This is the first comprehensive clinical study demonstrating the applicability of the LAMP assay for a rapid and reliable molecular diagnosis of VL and PKDL.