Evaluation of a Point-of-Need Molecular Diagnostic Tool Coupled with Rapid DNA Extraction Methods for Visceral Leishmaniasis.

A rapid, cost-effective, and simple nucleic acid isolation technique coupled with a point-of-need DNA amplification assay is a desirable goal for programmatic use. For diagnosis of Visceral Leishmaniasis (VL), Recombinase Polymerase Amplification (RPA) rapid tests for the detection of Leishmania DNA are versatile and have operational advantages over qPCR. To facilitate the delivery of the RPA test at point-of-need for VL diagnosis, we compared two rapid DNA extraction methods, SwiftDx (SX) and an in-house Boil and Spin (BS) method, coupled with RPA amplification, versus more widely used methods for DNA extraction and amplification, namely Qiagen (Q) kits and qPCR, respectively. A total of 50 confirmed VL patients and 50 controls, matched for age and gender, were recruited from Mymensingh, Bangladesh, a region highly endemic for VL. Blood samples were collected from each participant and DNA was extracted using Q, SX and BS methods. Following DNA extraction, qPCR and RPA assays were performed to detect L. donovani in downstream analysis. No significant differences in sensitivity of the RPA assay were observed between DNA extraction methods, 94.00% (95% CI: 83.45-98.75%), 90% (95% CI: 78.19-96.67%), and 88% (95% CI: 75.69-95.47%) when using Q, SX, and BS, respectively. Similarly, using qPCR, no significant differences in sensitivity were obtained when using Q or SX for DNA extraction, 94.00% (95% CI: 83.45-98.75%) and 92.00% (80.77-97.78%), respectively. It is encouraging that RPA and qPCR showed excellent agreement (k: 0.919-0.980) when different extraction methods were used and that the DNA impurities using BS had no inhibitory effect on the RPA assay. Furthermore, significantly higher DNA yields were obtained using SX and BS versus Q; however, a significantly higher parasite load was detected using qPCR when DNA was extracted using Q versus SX. Considering the cost, execution time, feasibility, and performance of RPA assay, rapid extraction methods such as the Boil and Spin technique appear to have the potential for implementation in resource-limited endemic settings. Further clinical research is warranted prior to broader application.

Assessment of treatment outcomes of visceral leishmaniasis (VL) treated cases and impact of COVID-19 on VL management and control services in Bangladesh.

BACKGROUND: COVID-19 has largely impacted the management of Visceral leishmaniasis (VL), like several other Neglected Tropical Diseases. The impact was particularly evident in Lower and Middle-Income countries where the already inadequate healthcare resources were diverted to managing the COVID-19 pandemic. Bangladesh achieved the elimination target for VL in 2016. To sustain this success, early diagnosis and treatment, effective vector control, and periodic surveillance are paramount. However, the specific control measures for VL in Bangladesh that were hampered during COVID-19 and their extent are unknown. METHODS: This study aimed at identifying the gaps and challenges in the follow-up of treated VL patients by interviewing both the treated VL cases and their health service providers. We followed VL cases treated between 2019 and 2020 in five VL endemic subdistricts (upazilas) both retrospectively and prospectively to monitor clinical improvement, relapse, or other consequences. Moreover, interviews were conducted with the health service providers to assess the impact of COVID-19 on VL case detection, treatment, reporting, vector control operations, and logistic supply chain management. RESULTS: There was no added delay for VL diagnosis; however, VL treatment initiation and reporting time increased almost two-fold due to COVID-19. Indoor Residual Spraying activity was significantly hampered due to a shortage of insecticides. Out of 44 enrolled and treated VL patients, two relapsed (4.5 %), two developed Para Kala-Azar Dermal Leishmaniasis (4.5 %), and three (6.8 %) Post Kala-Azar Dermal Leishmaniasis (PKDL). The health service providers highlighted patients` unwillingness to visit the hospital, financial constraints, and distance from the hospitals as the main reasons for missed follow-up visits (20.5 %). Building good communication in the community, awareness schemes, and incentive-based approaches were suggested as possible solutions to mitigate these problems. CONCLUSION: Long-term follow-up is required for the early detection and management of VL relapse and PKDL cases. Effective vector control measures, capacity development, and identification of new VL hotspots are pivotal in the VL endemic regions to sustain the elimination goal.

Gauging the skin resident Leishmania parasites through a loop mediated isothermal amplification (LAMP) assay in post-kala-azar dermal leishmaniasis.

Despite the availability of highly sensitive polymerase chain reaction (PCR)-based methods, the dearth of remotely deployable diagnostic tools circumvents the early and accurate detection of individuals with post-kala-azar dermal leishmaniasis (PKDL). Here, we evaluate a design-locked loop-mediated isothermal amplification (LAMP) assay to diagnose PKDL. A total of 76 snip-skin samples collected from individuals with probable PKDL (clinical presentation and a positive rK39 rapid diagnostic test (RDT)) were assessed by microscopy, qPCR, and LAMP. An equal number of age and sex-matched healthy controls were included to determine the specificity of the LAMP assay. The LAMP assay with a Qiagen DNA extraction (Q-LAMP) showed a promising sensitivity of 72.37% (95% CI: 60.91-82.01%) for identifying the PKDL cases. LAMP assay sensitivity declined when the DNA was extracted using a boil-spin method. Q-qPCR showed 68.42% (56.75-78.61%) sensitivity, comparable to LAMP and with an excellent agreement, whereas the microscopy exhibited a weak sensitivity of 39.47% (28.44-51.35%). When microscopy and/or qPCR were considered the gold standard, Q-LAMP exhibited an elevated sensitivity of 89.7% (95% CI: 78.83-96.11%) for detection of PKDL cases and Bayesian latent class modeling substantiated the excellent sensitivity of the assay. All healthy controls were found to be negative. Notwithstanding the optimum efficiency of the LAMP assay towards the detection of PKDL cases, further optimization of the boil-spin method is warranted to permit remote use of the assay.

Evaluation of molecular assays to detect Leishmania donovani in Phlebotomus argentipes fed on post-kala-azar dermal leishmaniasis patients.

BACKGROUND: Post-kala-azar dermal leishmaniasis (PKDL) caused by Leishmania donovani (LD) is a skin disorder that often appears after treatment of visceral leishmaniasis (VL) patients. PKDL patients are potential reservoirs of LD parasites, which can initiate a new epidemic of anthroponotic VL. Therefore, host infectiousness to its sand fly vector is a critical factor for transmission, and its accurate estimation can facilitate control strategies. At present, conventional microscopy serves as the reference method to detect parasites in its vector. However, low sensitivity of microscopy can be a limiting factor. METHODS: In this study, real-time quantitative PCR (LD-qPCR) and recombinase polymerase amplification (LD-RPA) assays were evaluated against microscopy for the detection of LD DNA extracted from live sand flies five days after controlled feeding on PKDL cases. RESULTS: The sensitivity of LD-qPCR and LD-RPA assays were found to be 96.43 and 100%, respectively, against microscopy for the selected fed sand flies (n = 28), and an absolute specificity of both molecular tools for apparently unfed sand flies (n = 30). While the proportion of infectious cases among 47 PKDL patients was estimated as 46.81% as defined by microscopic detection of LD in at least one fed sand fly per case, LD-RPA assay evaluation of only the microscopy negative sand flies fed to those 47 PKDL cases estimated an even greater proportion of infectious cases (51.06%). In overall estimation of the infectious cases in retrospective manner, discordance in positivity rate was observed (p < 0.05) between LD-RPA (59.57%) assay and microscopy (46.81%), while LD-RPA had slightly better positivity rate than LD-qPCR (55.32%) as well. CONCLUSIONS: Considering the sensitivity, cost, detection time, and field applicability, RPA assay can be considered as a promising single molecular detection tool for investigations pertaining to LD infections in sand flies and/or host infectiousness in PKDL, while it can also be useful in confirmation of microscopy negative sand fly samples.

Evaluation of Loopamp™ Leishmania Detection Kit and Leishmania Antigen ELISA for Post-Elimination Detection and Management of Visceral Leishmaniasis in Bangladesh.

With reduced prevalence of visceral leishmaniasis (VL) in the Indian subcontinent (ISC), direct and field deployable diagnostic tests are needed to implement an effective diagnostic and surveillance algorithm for post-elimination VL control. In this regard, here we investigated the diagnostic efficacies of a loop-mediated isothermal amplification (LAMP) assay (Loopamp™ Leishmania Detection Kit, Eiken Chemical CO., Ltd, Japan), a real-time quantitative PCR assay (qPCR) and the Leishmania antigen ELISA (CLIN-TECH, UK) with different sampling techniques and evaluated their prospect to incorporate into post-elimination VL control strategies. Eighty clinically and rK39 rapid diagnostic test confirmed VL cases and 80 endemic healthy controls were enrolled in the study. Peripheral blood and dried blood spots (DBS) were collected from all the participants at the time of diagnosis. DNA was extracted from whole blood (WB) and DBS via silica columns (QIAGEN) and boil & spin (B&S) methods and tested with qPCR and Loopamp. Urine was collected from all participants at the time of diagnosis and was directly subjected to the Leishmania antigen ELISA. 41 patients were followed up and urine samples were collected at day 30 and day 180 after treatment and ELISA was performed. The sensitivities of the Loopamp-WB(B&S) and Loopamp-WB(QIA) were 96.2% (95% CI 89·43-99·22) and 95% (95% CI 87·69-98·62) respectively. The sensitivity of Loopamp-DBS(QIA) was 85% (95% CI 75·26- 92·00). The sensitivities of the qPCR-WB(QIA) and qPCR-DBS(QIA) were 93.8% (95% CI 86·01-97·94) and 72.5% (95% CI 61·38-81·90) respectively. The specificity of all molecular assays was 100%. The sensitivity and specificity of the Leishmania antigen ELISA were 97.5% (95% CI 91·47-99·70) and 91.95% (95% CI 84·12-96·70) respectively. The Leishmania antigen ELISA depicted clinical cure at day 180 in all the followed-up cases. Efficacy and sustainability identify the Loopamp-WB(B&S) and the Leishmania antigen ELISA as promising and minimally invasive VL diagnostic tools to support VL diagnostic and surveillance activities respectively in the post-elimination era.

Evaluation of Rapid Extraction Methods Coupled with a Recombinase Polymerase Amplification Assay for Point-of-Need Diagnosis of Post-Kala-Azar Dermal Leishmaniasis.

To detect Post-kala-azar leishmaniasis (PKDL) cases, several molecular methods with promising diagnostic efficacy have been developed that involve complicated and expensive DNA extraction methods, thus limiting their application in resource-poor settings. As an alternative, we evaluated two rapid DNA extraction methods and determined their impact on the detection of the parasite DNA using our newly developed recombinase polymerase amplification (RPA) assay. Skin samples were collected from suspected PKDL cases following their diagnosis through national guidelines. The extracted DNA from three skin biopsy samples using three different extraction methods was subjected to RPA and qPCR. The qPCR and RPA assays exhibited highest sensitivities when reference DNA extraction method using Qiagen (Q) kit was followed. In contrast, the sensitivity of the RPA assay dropped to 76.7% and 63.3%, respectively, when the boil & spin (B&S) and SpeedXtract (SE) rapid extraction methods were performed. Despite this compromised sensitivity, the B&S-RPA technique yielded an excellent agreement with both Q-qPCR (k = 0.828) and Q-RPA (k = 0.831) techniques. As expected, the reference DNA extraction method was found to be superior in terms of diagnostic efficacy. Finally, to apply the rapid DNA extraction methods in resource-constrained settings, further methodological refinement is warranted to improve DNA yield and purity through rigorous experiments.

Imported cutaneous leishmaniasis: molecular investigation unveils Leishmania major in Bangladesh.

BACKGROUND: The main clinical forms of leishmaniasis in Bangladesh are visceral leishmaniasis and post-kala-azar dermal leishmaniasis, which are caused by Leishmania donovani. Imported cutaneous leishmaniasis (CL) is emerging globally due mainly to increased human mobility. In recent years, several imported CL cases have also been reported in Bangladesh. Sporadic atypical cases of CL can be challenging for diagnosis and clinical management, while occurrence of infection on a frequent basis can be alarming. We report of a case of a Bangladeshi temporary-migrant worker who, upon return, presented development of skin lesions that are characteristic of CL. METHODS: A serum sample was collected and tested with an rK39 immunochromatographic test. Nucleic acid from skin biopsy derived culture sample was extracted and screened with a real-time PCR assay which targets the conserved REPL repeat region of L. donovani complex. The internal transcribed spacer 2 region of the ribosomal RNA gene cluster was amplified and sequenced. RESULTS: The suspect had a history of travel in both CL and VL endemic areas and had a positive rK39 test result. Based on clinical presentation, travel history and demonstration of the parasite in the skin biopsy, CL was diagnosed and the patient underwent a combination therapy with Miltefosine and liposomal amphotericin B. While typical endemic species were not detected, we identified Leishmania major, a species that, to our knowledge, has never been reported in Bangladesh. CONCLUSIONS: Proper monitoring and reporting of imported cases should be given careful consideration for both clinical and epidemiological reasons. Molecular tests should be performed in diagnosis to avoid dilemma, and identification of causative species should be prioritized.

Relationship of Serum Antileishmanial Antibody With Development of Visceral Leishmaniasis, Post-kala-azar Dermal Leishmaniasis and Visceral Leishmaniasis Relapse.

INTRODUCTION: To sustain the achievement of kala-azar elimination program (KEP), early detection and treatment of the visceral leishmaniasis (VL) cases and associated modalities such as treatment failure (TF), relapse VL (RVL), and Post-kala-azar dermal leishmaniasis (PKDL) is the cornerstone. A predictive biomarker for VL development and related complications could also play a crucial role in curtailing disease incidence and transmission. Investigations to find a biomarker with prospective capabilities are, however, scarce. Using samples and known clinical outcomes generated within two previous longitudinal cohort studies, we aimed to determine if fluctuations in serum anti-rK39 antibody levels could provide such predictive value. MATERIALS AND METHODS: Serum samples collected at four different time points (Baseline, 12, 18, and 24 months) from 16 patients who had developed VL within the monitoring period and 15 of their asymptomatic healthy controls counterparts were investigated. To investigate potential prediction of VL related complications, serum samples of 32 PKDL, 10 RVL, 07 TF, and 38 cured VL from a single dose AmBisome trial were analyzed. Of this second panel, all patients were monitored for 5 years and sera were collected at four time points (Baseline then 1, 6, and 12 months after treatment). The level of anti-rK39 antibodies in archived samples was measured by a semi-quantitative ELISA. RESULTS: The mean antibody level was significantly higher in VL patients compared to their asymptomatic healthy counterparts at each time point. Likewise, we observed a trend toward elevations in antibody levels for PKDL, RVL, TF relative to the reducing levels observed in cured VL. Receiver operating characteristic (ROC) analysis found a promising predictive power of rK39 antibody levels to reveal progression from asymptomatic Leishmania donovani infection stage to VL, defined as 87.5% sensitive and 95% specific. Following treatment, rk39 antibody notably showed 100% sensitivity and 95% specificity in predicting TF. CONCLUSION: Our data indicate that the relative quantity of serum anti-rK39 antibody has promise within either a predictive or prognostic algorithm for VL and VL-related modalities. These could enable VL control programs to implement more effective measures to eliminate the disease. Further research is, however, imperative to standardize the rK39 antibody ELISA between sites prior to broader use.

Quantifying the Infectiousness of Post-Kala-Azar Dermal Leishmaniasis Toward Sand Flies.

BACKGROUND: On the Indian subcontinent, visceral leishmaniasis (VL) incidence is on track to reach elimination goals by 2020 in nearly all endemic districts. Although not included in official targets, previous data suggest post-kala-azar dermal leishmaniasis (PKDL) patients can act as an infection reservoir. METHODS: We conducted xenodiagnosis on 47 PKDL patients and 15 VL patients using laboratory-reared Phlebotomus argentipes. In direct xenodiagnosis, flies were allowed to feed on the patient's skin for 15 minutes. For indirect xenodiagnosis, flies were fed through a membrane on the patient's blood. Five days later, blood-fed flies were dissected and examined by microscopy and/or polymerase chain reaction (PCR). A 3-mm skin snip biopsy (PKDL) or venous blood (VL) was processed by quantitative PCR. RESULTS: Twenty-seven PKDL patients (57.4%) had positive results by direct and/or indirect xenodiagnosis. Direct was significantly more sensitive than indirect xenodiagnosis (55.3% vs 6.4%, P < .0001). Those with positive xenodiagnosis had median skin parasite loads >1 log10 unit higher than those with negative results (2.88 vs 1.66, P < .0001). In a multivariable model, parasite load, nodular lesions, and positive skin microscopy were significantly associated with positive xenodiagnosis. Blood parasite load was the strongest predictor for VL. Compared to VL, nodular PKDL was more likely and macular PKDL less likely to result in positive xenodiagnosis, but neither difference reached statistical significance. CONCLUSIONS: Nodular and macular PKDL, and VL, can be infectious to sand flies. Active PKDL case detection and prompt treatment should be instituted and maintained as an integral part of VL control and elimination programs.

Evaluation of Real-time PCR for Diagnosis of Post-Kala-azar Dermal Leishmaniasis in Endemic Foci of Bangladesh.

BACKGROUND: Post-kala-azar dermal leishmaniasis (PKDL) is a sequel to visceral leishmaniasis (VL), which is found in VL-endemic countries including Bangladesh. Because of these enigmatic cases, the success of the National Kala-azar Elimination Program is under threat. To date, diagnostic methods for PKDL cases in endemic regions have been limited to clinical examination and rK39 test or microscopy, and a suitable and accurate alternative method is needed. In this study, we investigated the application of real-time polymerase chain reaction (PCR) as a potential method for diagnosis of PKDL in comparison with microscopy. METHODS: Ninety-one suspected macular PKDL cases from Mymensingh district, Bangladesh, were enrolled in the study after diagnosis by clinical examination and an rK39 strip test. All of them responded after completion of the treatment with miltefosine. During enrollment, a skin biopsy was done for each patient, and both microscopy and real-time PCR were performed for detection and quantification of Leishmania donovan body (LDB) and LD DNA, respectively. RESULTS: Real-time PCR detected 83 cases among all suspected PKDL patients, with an encouraging sensitivity of 91.2% (83.4%-96.1%), whereas microscopy showed 50.6% (39.9%-61.2%) sensitivity. Among all suspected PKDL cases, 42 cases were positive in both microscopy and qPCR, whereas 41 cases were detected as positive through qPCR only. CONCLUSIONS: This study provides evidence that real-time PCR is a promising tool for diagnosis of PKDL in endemic regions. In addition to diagnosis, the quantitative ability of this method could be further exploited for after-treatment prognosis and cure assessment of PKDL cases.

Case Report: Treatment of Widespread Nodular Post kala-Azar Dermal Leishmaniasis with Extended-Dose Liposomal Amphotericin B in Bangladesh: A Series of Four Cases.

Post kala-azar dermal leishmaniasis (PKDL) is a skin manifestation which usually appears after visceral leishmaniasis. It is now proved that PKDL patients serve as a reservoir for anthropometric leishmanial transmission. Hence, to achieve the kala-azar elimination target set by the World Health Organization in the Indian Subcontinent, PKDL cases should be given priority. The goal of treatment for PKDL should be early reepithelizlization and rapid cure, but unfortunately this has been difficult to achieve, especially for patients with severe lesions. Therefore, we describe here four cases of PKDL who had widespread nodular and macular lesions and were treated with two cycles of LAmB doses with 20 mg/kg body weight divided into four equal doses (each dose contains 5 mg/kg) administered every alternate day. This treatment schedule achieved 100% treatment success with the minimal safety concern.