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

2021 ◽  
Vol 11 ◽  
Author(s):  
Faria Hossain ◽  
Albert Picado ◽  
Sophie I. Owen ◽  
Prakash Ghosh ◽  
Rajashree Chowdhury ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Control Strategies ◽  
Indian Subcontinent ◽  
Lamp Assay ◽  
Dried Blood Spots ◽  
Diagnostic Tools ◽  
Real Time Quantitative Pcr ◽  
Antigen Elisa ◽  
Eiken Chemical ◽  
Quantitative Pcr Assay

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.

scholarly journals Detection of asymptomatic Leishmania infection in Bangladesh by novel antigen and antibody diagnostic tools shows strong association with PKDL patients

2020 ◽  
Author(s):  
Sophie Owen ◽  
Faria Hossain ◽  
Prakash Ghosh ◽  
Rajashree Chowdhury ◽  
Md. Sakhawat Hossain ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Quantitative Polymerase Chain Reaction ◽  
Asymptomatic Infection ◽  
Diagnostic Tools ◽  
Leishmania Infection ◽  
Active Infection ◽  
Non Invasive ◽  
Antigen Elisa ◽  
Disease Reservoirs ◽  
Index Cases

AbstractBackgroundAsymptomatic Leishmania infections outnumber clinical infections on the Indian sub-continent (ISC) where disease reservoirs are anthroponotic. Diagnostics which detect active asymptomatic infection, which are suitable for monitoring and surveillance, may be of benefit to the visceral leishmaniasis (VL) elimination campaign on the ISC.Methodology/Principal FindingsQuantitative polymerase chain reaction (qPCR), loop mediated isothermal amplification (LAMP), the direct agglutination test (DAT), and the Leishmania antigen ELISA were carried out on blood and urine samples collected from 720 household and neighbouring contacts of 276 VL and post kala-azar dermal leishmaniasis (PKDL) index cases, with no symptoms or history of VL and PKDL, in endemic regions of Bangladesh between September 2016 and March 2018. Of the 720 contacts of index cases, asymptomatic infection was detected in 69 (9.6%) participants by a combination of qPCR (1.0%), LAMP (2.1%), DAT (3.9%), and Leishmania antigen ELISA (3.3%). Only 1 (0.1%) participant was detected positive by all 4 diagnostic tests. Poor agreement between tests was calculated using Cohen’s kappa (k) statistics, however the Leishmania antigen ELISA and DAT in combination capture all participants positive by more than one test. We find strong evidence for association between the index case being a PKDL case (OR 1.94, p = 0.009), specifically macular PKDL (OR 2.12, p = 0.004) and being positive for at least one of the four tests.Conclusions/SignificanceLeishmania antigen ELISA detects active asymptomatic infection, requires a non-invasive sample, and therefore may be of benefit for monitoring transmission and surveillance in an elimination setting in combination with serology. Development of an antigen detection test in RDT format would be of benefit to the elimination campaign.Author summaryInfection with the parasite Leishmania donovani can lead to an asymptomatic infection with only around 5% of asymptomatics converting to visceral leishmaniasis the clinical manifestation of the infection. Serological assays detect anti-Leishmania antibodies and therefore cannot distinguish between past and active infection. Molecular assays and those which detect Leishmania antigens detect active infection. Since the signing of a memorandum of understanding in 2005, visceral leishmaniasis has been targeted for elimination in India, Nepal and Bangladesh. In an elimination setting such as Bangladesh, where disease reservoirs are anthroponotic, a relatively simple test such as the Leishmania antigen ELISA which requires a non-invasive urine sample, may be of benefit in combination with serology for surveillance and monitoring of Leishmania transmission. Development of an antigen test into a field compatible rapid diagnostic test would be of further benefit to the elimination campaign.

scholarly journals Detection of asymptomatic Leishmania infection in Bangladesh by antibody and antigen diagnostic tools shows an association with post–kala-azar dermal leishmaniasis (PKDL) patients

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Sophie I. Owen ◽  
Faria Hossain ◽  
Prakash Ghosh ◽  
Rajashree Chowdhury ◽  
Md. Sakhawat Hossain ◽  
...  
Keyword(s):  
Indian Subcontinent ◽  
Quantitative Polymerase Chain Reaction ◽  
Strong Association ◽  
Asymptomatic Infection ◽  
Diagnostic Tools ◽  
Leishmania Infection ◽  
Kala Azar ◽  
Antigen Elisa ◽  
History Of ◽  
Index Cases

Abstract Background Asymptomatic Leishmania infections outnumber clinical infections on the Indian subcontinent (ISC), where disease reservoirs are anthroponotic. Diagnostics which detect active asymptomatic infection, which are suitable for monitoring and surveillance, may be of benefit to the visceral leishmaniasis (VL) elimination campaign on the ISC. Methods Quantitative polymerase chain reaction (qPCR), loop-mediated isothermal amplification (LAMP), and the direct agglutination test (DAT) were carried out on blood samples, and the Leishmania antigen ELISA was carried out on urine samples collected from 720 household and neighbouring contacts of 276 VL and post–kala-azar dermal leishmaniasis (PKDL) index cases, with no symptoms or history of VL or PKDL, in endemic regions of Bangladesh between September 2016 and March 2018. Results Of the 720 contacts of index cases, asymptomatic infection was detected in 69 (9.6%) participants by a combination of qPCR (1.0%), LAMP (2.1%), DAT (3.9%), and Leishmania antigen ELISA (3.3%). Only one (0.1%) participant was detected positive by all four diagnostic tests. Poor agreement between tests was calculated using Cohen’s kappa (κ) statistics; however, the Leishmania antigen ELISA and DAT in combination captured all participants as positive by more than one test. We find evidence for a moderately strong association between the index case being a PKDL case (OR 1.94, p = 0.009), specifically macular PKDL (OR 2.12, p = 0.004), and being positive for at least one of the four tests. Conclusions Leishmania antigen ELISA on urine detects active asymptomatic infection, requires a non-invasive sample, and therefore may be of benefit for monitoring transmission and surveillance in an elimination setting in combination with serology. Development of an antigen detection test in a rapid diagnostic test (RDT) format would be of benefit to the elimination campaign.

scholarly journals Loop-Mediated Isothermal Amplification Allows Rapid, Simple and Accurate Molecular Diagnosis of Human Cutaneous and Visceral Leishmaniasis Caused by Leishmania infantum When Compared to PCR

2021 ◽  
Vol 9 (3) ◽  
pp. 610
Author(s):  
Ana Victoria Ibarra-Meneses ◽  
Carmen Chicharro ◽  
Carmen Sánchez ◽  
Emilia García ◽  
Sheila Ortega ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Real Time ◽  
Parasite Load ◽  
Isothermal Amplification ◽  
Excellent Correlation ◽  
Loop Mediated Isothermal Amplification ◽  
Real Time Quantitative Pcr ◽  
Specific Amplification ◽  
Polymerase Chain ◽  
Eiken Chemical

Loop-mediated isothermal amplification allows the rapid, sensitive and specific amplification of DNA without complex and expensive equipment. We compared the diagnostic performance of Loopamp™ Leishmania Detection Kit (Eiken Chemical Co., Ltd., Tokyo, Japan) with conventional and real-time polymerase chain reaction (PCR) for human cutaneous and visceral leishmaniasis caused by L. infantum. A total of 230 DNA samples from cutaneous (CL) and visceral (VL) leishmaniasis cases and controls from Spain, characterized by Leishmania nested PCR (LnPCR) were tested by: (i) the Loopamp™ Leishmania Detection Kit (Loopamp), run on Genie III real-time fluorimeter (OptiGene, UK); and (ii) real-time quantitative PCR (qPCR). The Loopamp test returned 98.8% (95% confidence interval—CI: 96.0–100.00) sensitivity and specificity of 97.7% (95% CI: 92.2–100) on VL samples, and 100% (95% CI: 99.1–100) sensitivity and 100.0% (95% CI: 98.8–100.0) specificity on CL samples. The Loopamp time-to-positivity (Tp) obtained by real-time fluorimetry showed excellent concordance (C = 97.91%) and strong correlation (r = 0.799) with qPCR’s cycle threshold (Ct). The performance of Loopamp is comparable to that of LnPCR and qPCR in the diagnosis of cutaneous and visceral leishmaniasis due to L. infantum. The excellent correlation between the Tp and Ct should be further investigated to determine the accuracy of Loopamp to quantify parasite load in tissues.

scholarly journals Evaluation of qPCR on blood and skin microbiopsies, peripheral blood buffy coat smear, and urine antigen ELISA for diagnosis and test of cure for visceral leishmaniasis in HIV-coinfected patients in India: a prospective cohort study

BMJ Open ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. e042519
Author(s):  
Sophie I Owen ◽  
Sakib Burza ◽  
Shiril Kumar ◽  
Neena Verma ◽  
Raman Mahajan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Visceral Leishmaniasis ◽  
Hiv Infection ◽  
Peripheral Blood ◽  
Medical Science ◽  
Bone Marrow Aspiration ◽  
Buffy Coat ◽  
Tropical Medicine ◽  
Urine Antigen ◽  
Antigen Elisa

IntroductionHIV coinfection presents a challenge for diagnosis of visceral leishmaniasis (VL). Invasive splenic or bone marrow aspiration with microscopic visualisation of Leishmania parasites remains the gold standard for diagnosis of VL in HIV-coinfected patients. Furthermore, a test of cure by splenic or bone marrow aspiration is required as patients with VL-HIV infection are at a high risk of treatment failure. However, there remain financial, implementation and safety costs to these invasive techniques which severely limit their use under field conditions.Methods and analysisWe aim to evaluate blood and skin qPCR, peripheral blood buffy coat smear microscopy and urine antigen ELISA as non-invasive or minimally invasive alternatives for diagnosis and post-treatment test of cure for VL in HIV-coinfected patients in India, using a sample of 91 patients with parasitologically confirmed symptomatic VL-HIV infection.Ethics and disseminationEthical approval for this study has been granted by The Liverpool School of Tropical Medicine, The Institute of Tropical Medicine in Antwerp, the University of Antwerp and the Rajendra Memorial Research Institute of Medical Science in Patna. Any future publications will be published in open access journals.Trial registration numberCTRI/2019/03/017908.

scholarly journals 630. A 5-mRNA host response whole-blood classifier trained using patients with non-COVID-19 viral infections accurately predicts severity of COVID-19

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S375-S376
Author(s):  
ljubomir Buturovic ◽  
Purvesh Khatri ◽  
Benjamin Tang ◽  
Kevin Lai ◽  
Win Sen Kuan ◽  
...  
Keyword(s):  
Logistic Regression ◽  
Respiratory Failure ◽  
Viral Infections ◽  
Lamp Assay ◽  
Training Data ◽  
Diagnostic Tools ◽  
Severe Respiratory Failure ◽  
Proof Of Concept ◽  
Risk Of Death ◽  
Qpcr Platform

Abstract Background While major progress has been made to establish diagnostic tools for the diagnosis of SARS-CoV-2 infection, determining the severity of COVID-19 remains an unmet medical need. With limited hospital resources, gauging severity would allow for some patients to safely recover in home quarantine while ensuring sicker patients get needed care. We discovered a 5 host mRNA-based classifier for the severity of influenza and other acute viral infections and validated the classifier in COVID-19 patients from Greece. Methods We used training data (N=705) from 21 retrospective clinical studies of influenza and other viral illnesses. Five host mRNAs from a preselected panel were applied to train a logistic regression classifier for predicting 30-day mortality in influenza and other viral illnesses. We then applied this classifier, with fixed weights, to an independent cohort of subjects with confirmed COVID-19 from Athens, Greece (N=71) using NanoString nCounter. Finally, we developed a proof-of-concept rapid, isothermal qRT-LAMP assay for the 5-mRNA host signature using the QuantStudio 6 qPCR platform. Results In 71 patients with COVID-19, the 5 mRNA classifier had an AUROC of 0.88 (95% CI 0.80-0.97) for identifying patients with severe respiratory failure and/or 30-day mortality (Figure 1). Applying a preset cutoff based on training data, the 5-mRNA classifier had 100% sensitivity and 46% specificity for identifying mortality, and 88% sensitivity and 68% specificity for identifying severe respiratory failure. Finally, our proof-of-concept qRT-LAMP assay showed high correlation with the reference NanoString 5-mRNA classifier (r=0.95). Figure 1. Validation of the 5-mRNA classifier in the COVID-19 cohort. (A) Expression of the 5 genes used in the logistic regression model in patients with (red) and without (blue) mortality. (B) The 5-mRNA classifier accurately distinguishes non-severe and severe patients with COVID-19 as well as those at risk of death. Conclusion Our 5-mRNA classifier demonstrated very high accuracy for the prediction of COVID-19 severity and could assist in the rapid, point-of-impact assessment of patients with confirmed COVID-19 to determine level of care thereby improving patient management and healthcare burden. Disclosures ljubomir Buturovic, PhD, Inflammatix Inc. (Employee, Shareholder) Purvesh Khatri, PhD, Inflammatix Inc. (Shareholder) Oliver Liesenfeld, MD, Inflammatix Inc. (Employee, Shareholder) James Wacker, n/a, Inflammatix Inc. (Employee, Shareholder) Uros Midic, PhD, Inflammatix Inc. (Employee, Shareholder) Roland Luethy, PhD, Inflammatix Inc. (Employee, Shareholder) David C. Rawling, PhD, Inflammatix Inc. (Employee, Shareholder) Timothy Sweeney, MD, Inflammatix, Inc. (Employee)

scholarly journals Development of a loop-mediated isothermal amplification (LAMP) assay for the identification of the invasive wood borer Aromia bungii (Coleoptera: Cerambycidae) from frass

3 Biotech ◽  
2021 ◽  
Vol 11 (2) ◽  
Author(s):  
Domenico Rizzo ◽  
Nicola Luchi ◽  
Daniele Da Lio ◽  
Linda Bartolini ◽  
Francesco Nugnes ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Isothermal Amplification ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Loop Mediated Isothermal Amplification ◽  
Larval Stages ◽  
Longhorn Beetle ◽  
Rapid Monitoring ◽  
Major Pest ◽  
Wood Borer

AbstractThe red-necked longhorn beetle Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it is a major pest of cultivated and ornamental species of the genus Prunus. Morphological or molecular discrimination of adults or larval specimens is required to identify this invasive wood borer. However, recovering larval stages of the pest from trunks and branches causes extensive damage to plants and is timewasting. An alternative approach consists in applying non-invasive molecular diagnostic tools to biological traces (i.e., fecal pellets, frass). In this way, infestations in host plants can be detected without destructive methods. This paper presents a protocol based on both real-time and visual loop-mediated isothermal amplification (LAMP), using DNA of A. bungii extracted from fecal particles in larval frass. Laboratory validations demonstrated the robustness of the protocols adopted and their reliability was confirmed performing an inter-lab blind panel. The LAMP assay and the qPCR SYBR Green method using the F3/B3 LAMP external primers were equally sensitive, and both were more sensitive than the conventional PCR (sensitivity > 103 to the same starting matrix). The visual LAMP protocol, due to the relatively easy performance of the method, could be a useful tool to apply in rapid monitoring of A. bungii and in the management of its outbreaks.

scholarly journals Investigating the dynamics of Leishmania antigen in the urine of patients with visceral leishmaniasis: a pilot study

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 1514
Author(s):  
Prakash Ghosh ◽  
Israel Cruz ◽  
Albert Picado ◽  
Thomas Edwards ◽  
Md. Anik Ashfaq Khan ◽  
...  
Keyword(s):  
Pilot Study ◽  
Visceral Leishmaniasis ◽  
Treatment Monitoring ◽  
Urine Samples ◽  
Urinary Antigen ◽  
Antigen Concentration ◽  
Elisa Kit ◽  
Time Points ◽  
Non Invasive ◽  
Antigen Elisa

Background: Detection of Leishmania antigens in the urine provides a non-invasive means of diagnosis and treatment monitoring of cases of visceral leishmaniasis (VL). Leishmania antigen load in the urine may vary between different time-points within a day, thus influencing the performance of antigen-detection tests. Methods: We investigated the dynamics of Leishmania antigen in urine collected at three different time points (08:00, 12:00 and 16:00 hours). All urine samples collected were tested with the Leishmania Antigen ELISA (VL ELISA) kit, produced by Kalon Biological Ltd., UK. Results: The median concentration of Leishmania antigen in urine collected at 08:00 (2.7 UAU-urinary antigen units/ml) was higher than at 12:00 (1.7 UAU/ml) and at 16:00 (1.9 UAU/ml). These differences were found to be statistically significant (08:00 vs. 12:00, p=0.011; 08:00 vs. 16:00, p=0.041). Conclusion: This pilot study indicates that the Leishmania antigen concentration is higher in urine samples collected in the morning, which has important implications when the VL ELISA kit or other tests to detect Leishmania antigen in urine are used for diagnosis of VL and treatment monitoring.

scholarly journals Visceral leishmaniasis (Kala-azar) in Qom Province, Iran: Report of two cases

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 1371
Author(s):  
Leyli Zanjirani Farahani ◽  
Abedin Saghafipour ◽  
Mehdi Mohebali ◽  
Behnaz Akhoundi ◽  
Hedayatollah Raufi
Keyword(s):  
Visceral Leishmaniasis ◽  
Rural Areas ◽  
Control Strategies ◽  
Sand Fly ◽  
Recurrent Fever ◽  
Kala Azar ◽  
Periodic Monitoring ◽  
History Of ◽  
The Common

Visceral leishmaniasis (VL) is a fatal parasitic zoonotic worldwide disease, which transmits to humans by the infected Phlebotomine sand fly bite. The common form of VL in Iran is the Mediterranean type with the causative agent of Leishmania infantum, whose main reservoirs are stray and domesticated dogs. The disease has several endemic foci in Iran, mostly seen among children under the age of 10, living in rural areas and nomadic tribes. The first cases of Kala-Azar in Qom province, central Iran, were reported in the year 2001, from the villages of Ghahan district. After conducting VL control strategies in the area, no new cases of the disease had been reported until recently. The cases described here are two 2-year-old girls, living in the urban parts of Qom province, one of whom did not have a history of traveling to known endemic areas of the disease. The patients were admitted to hospital in 2016-2017, complaining from recurrent fever with unrecognized reason, associated with decreased appetite and weight loss. Disease follow-up demonstrated anemia and splenomegaly, which led to diagnosis of VL, and both patients are now fully recovered. VL was presumed to be controlled in Qom province but the present cases indicate that possible VL existence remains in the region. Therefore, urgent studies and periodic monitoring are needed to identify potential reservoirs of VL in the area.

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