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

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          Abstract

          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.

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          Post-kala-azar dermal leishmaniasis.

          E Zijlstra, A. M. Musa, E A G Khalil … (2003)
          Post-kala-azar dermal leishmaniasis (PKDL) is a complication of visceral leishmaniasis (VL); it is characterised by a macular, maculopapular, and nodular rash in a patient who has recovered from VL and who is otherwise well. The rash usually starts around the mouth from where it spreads to other parts of the body depending on severity. It is mainly seen in Sudan and India where it follows treated VL in 50% and 5-10% of cases, respectively. Thus, it is largely restricted to areas where Leishmania donovani is the causative parasite. The interval at which PKDL follows VL is 0-6 months in Sudan and 2-3 years in India. PKDL probably has an important role in interepidemic periods of VL, acting as a reservoir for parasites. There is increasing evidence that the pathogenesis is largely immunologically mediated; high concentrations of interleukin 10 in the peripheral blood of VL patients predict the development of PKDL. During VL, interferon gamma is not produced by peripheral blood mononuclear cells (PBMC). After treatment of VL, PBMC start producing interferon gamma, which coincides with the appearance of PKDL lesions due to interferon-gamma-producing cells causing skin inflammation as a reaction to persisting parasites in the skin. Diagnosis is mainly clinical, but parasites can be seen by microscopy in smears with limited sensitivity. PCR and monoclonal antibodies may detect parasites in more than 80% of cases. Serological tests and the leishmanin skin test are of limited value. Treatment is always needed in Indian PKDL; in Sudan most cases will self cure but severe and chronic cases are treated. Sodium stibogluconate is given at 20 mg/kg for 2 months in Sudan and for 4 months in India. Liposomal amphotericine B seems effective; newer compounds such as miltefosine that can be administered orally or topically are of major potential interest. Although research has brought many new insights in pathogenesis and management of PKDL, several issues in particular in relation to control remain unsolved and deserve urgent attention.
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            NanoDrop microvolume quantitation of nucleic acids.

            Philippe Desjardins, Deborah Conklin (2010)
            Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation. The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results. The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 μL of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 μL or less are now possible using a dedicated microvolume fluorospectrometer. Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ μL to 15,000 ng/ μL with minimal consumption of sample.
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              A meta-analysis of the diagnostic performance of the direct agglutination test and rK39 dipstick for visceral leishmaniasis.

              François Chappuis, Marleen Boelaert, Luis Soto … (2006)
              To compare the performance of the direct agglutination test and rK39 dipstick for the diagnosis of visceral leishmaniasis. Medline, citation tracking, January 1986 to December 2004. Selection criteria Original studies evaluating the direct agglutination test or the rK39 dipstick with clinical visceral leishmaniasis as target condition; adequate reference classification; and absolute numbers of true positive, true negative, false positive, and false negative observations available or derivable from the data presented. 30 studies evaluating the direct agglutination test and 13 studies evaluating the rK39 dipstick met the inclusion criteria. The combined sensitivity estimates of the direct agglutination test and the rK39 dipstick were 94.8% (95% confidence interval 92.7% to 96.4%) and 93.9% (87.7% to 97.1%), respectively. Sensitivity seemed higher and more homogenous in the studies carried out in South Asia. Specificity estimates were influenced by the type of controls. In phase III studies carried out on patients with clinically suspected disease, the estimated specificity of the direct agglutination test was 85.9% (72.3% to 93.4%) and of the rK39 dipstick was 90.6% (66.8% to 97.9%). The diagnostic performance of the direct agglutination test and the rK39 dipstick for visceral leishmaniasis is good to excellent and seem comparable.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Trop Med Infect Dis
                Journal ID (iso-abbrev): Trop Med Infect Dis
                Journal ID (publisher-id): tropicalmed
                Title: Tropical Medicine and Infectious Disease
                Publisher: MDPI
                ISSN (Electronic): 2414-6366
                Publication date (Electronic): 05 June 2020
                Publication date Collection: June 2020
                Volume: 5
                Issue: 2
                Electronic Location Identifier: 95
                Affiliations
                [1 ]Nutrition and Clinical Service Division (NCSD), International Centre for Diarrhoeal Disease Research, Bangladesh, (icddr,b), Dhaka 1212, Bangladesh; r.chowdhury@ 123456icddrb.org (R.C.); anik.khan@ 123456icddrb.org (M.A.A.K.); faria.hossain@ 123456icddrb.org (F.H.); khaledul.faisal@ 123456icddrb.org (K.F.); rupen.nath@ 123456icddrb.org (R.N.); james.baker@ 123456icddrb.org (J.B.); shomik.maruf@ 123456icddrb.org (S.M.); debashis@ 123456icddrb.org (D.G.); utbabinrashid@ 123456icddrb.org (M.U.B.R.); din63d@ 123456icddrb.org (D.M.)
                [2 ]Institute of Animal Hygiene and Veterinary Public Health, University of Leipzig, D-04103 Leipzig, Germany; abdelwahed@ 123456gwdg.de
                [3 ]London School of Hygiene and Tropical Medicine, University of London, London WC1E 7HT, UK
                [4 ]Infection and Tropical Medicine, Mymensingh Medical College and Hospital (MMCH), Mymensingh 2200, Bangladesh; progganath@ 123456yahoo.com
                [5 ]Department of Cardiovascular and Thoracic Surgery, National Heart Foundation Hospital and Research Institute, Dhaka 1216, Bangladesh; mrashid1580@ 123456gmail.com
                [6 ]Host Directed Therapeutics (HDT) Bio Corp, Seattle, WA 98102, USA; malcolm.duthie@ 123456hdtbiocorp.com
                Author notes
                [* ]Correspondence: prakash.ghosh@ 123456icddrb.org ; Tel.: +880-1731855586
                [†]

                These authors contributed equally to this work.

                Author information
                https://orcid.org/0000-0003-2619-2445
                https://orcid.org/0000-0002-2530-9982
                Article
                Publisher ID: tropicalmed-05-00095
                DOI: 10.3390/tropicalmed5020095
                PMC ID: 7344569
                PubMed ID: 32517156
                SO-VID: 71047415-acd0-4904-aac0-278b59373d9a
                Copyright © © 2020 by the authors.
                License:

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 16 April 2020
                Date accepted : 20 May 2020
                Categories
                Subject: Article

                Keywords: post-kala-azar dermal leishmaniasis (pkdl),point-of-need diagnosis,dna extraction,recombinase polymerase amplification (rpa),real-time pcr
                Data availability:
                Keywords: post-kala-azar dermal leishmaniasis (pkdl), point-of-need diagnosis, dna extraction, recombinase polymerase amplification (rpa), real-time pcr

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