Sensitive and less invasive confirmatory diagnosis of visceral leishmaniasis in Sudan using loop-mediated isothermal amplification (LAMP)

<div><p>Background</p><p>Confirmatory diagnosis of visceral leishmaniasis (VL), as well as diagnosis of relapses and test of cure, usually requires examination by microscopy of samples collected by invasive means, such as splenic, bone marrow or lymph node aspirates. This causes discomfort to patients, with risks of bleeding and iatrogenic infections, and requires technical expertise. Molecular tests have great potential for diagnosis of VL using peripheral blood, but require well-equipped facilities and trained personnel. More user-friendly, and field-amenable options are therefore needed. One method that could meet these requirements is loop-mediated isothermal amplification (LAMP) using the Loopamp <i>Leishmania</i> Detection Kit, which comes as dried down reagents that can be stored at room temperature, and allows simple visualization of results.</p><p>Methodology/Principal findings</p><p>The Loopamp <i>Leishmania</i> Detection Kit (Eiken Chemical Co., Japan), was evaluated in the diagnosis of VL in Sudan. A total of 198 VL suspects were tested by microscopy of lymph node aspirates (the reference test), direct agglutination test-DAT (in house production) and rK28 antigen-based rapid diagnostic test (OnSite <i>Leishmania</i> rK39-Plus, CTK Biotech, USA). LAMP was performed on peripheral blood (whole blood and buffy coat) previously processed by: i) a direct boil and spin method, and ii) the QIAamp DNA Mini Kit (QIAgen). Ninety seven of the VL suspects were confirmed as cases by microscopy of lymph node aspirates. The sensitivity and specificity for each of the tests were: rK28 RDT 98.81% and 100%; DAT 88.10% and 78.22%; LAMP-boil and spin 97.65% and 99.01%; LAMP-QIAgen 100% and 99.01%.</p><p>Conclusions/Significance</p><p>Due to its simplicity and high sensitivity, rK28 RDT can be used first in the diagnostic algorithm for primary VL diagnosis, the excellent performance of LAMP using peripheral blood indicates that it can be also included in the algorithm for diagnosis of VL as a simple test when parasitological confirmatory diagnosis is required in settings that are lower than the reference laboratory, avoiding the need for invasive lymph node aspiration.</p></div

Identification of positive and negative LAMP results using blue LED light illumination with a visualization unit that is integral to the Loopamp LF-160 incubator.

<p>Identification of positive and negative LAMP results using blue LED light illumination with a visualization unit that is integral to the Loopamp LF-160 incubator.</p

Role of Loopamp <i>Leishmania</i> Detection Kit in the VL diagnostic algorithm.

<p>Role of Loopamp <i>Leishmania</i> Detection Kit in the VL diagnostic algorithm.</p

Details and summary of the diagnostic performance of different studies using LAMP for VL diagnosis compared to our study.

<p>Details and summary of the diagnostic performance of different studies using LAMP for VL diagnosis compared to our study.</p

Analytical sensitivity of LAMP assay.

<p>Top panel: Fluorescence observed with the naked eye from serial dilutions obtained from 3 different aliquots of DNA from <i>CL</i> Brener stock (DTU VI). Bottom panel: Fluorescence observed with the naked eye from serial dilutions obtained from 3 different aliquots of DNA from Sylvio X10 stock.The aliquots were expressed in fg/μL. A: 0; B: 1.0 x 10⁻³; C: 1.0 x 10⁻²; D: 1.0 x 10⁻¹, E: 1. NC: Non template control.</p

Evaluation of clinical specimens using LAMP assay.

<p>A. Visualization by Naked Eye: 1. positive control; 2. CCD6 Chronic Chagas Disease (case 6); 3. NI8: non-infected patient, (case 8); 4. AI-TxRID 2: acute infection of transplanted recipient from infected donor (case 2); 5. RCD 1: reactivated Chagas disease (case 1); 6. CCD1: chronic Chagas disease 1 (case 1); 7. CI 4: congenital Chagas disease (case 4); 8. negative control. B. Detection of LAMP reaction using Genie III Fluorimeter. 1: positive control; 2 to 7: clinical specimens indicated in A; 7: Negative control. The Y Axis denotes Fluorescence and X axis denotes Tt (time when fluorescence passes the threshold).</p

Inclusivity of LAMP assay tested in purified DNA samples from <i>T</i>. <i>cruzi</i> strains representative of the different discrete type units.

<p>Left panels: Visualization by the naked eye of <i>T</i>. <i>cruzi</i> DNA stocks representative of different DTUs. Tc I (A: 1.0 x10^-2 fg/test; B: 1.0 x10^-3 fg/test); Tc II (C: 2.5 fg/test; D: 2.5x10^-1 fg/test); Tc III (E: 7.5 x 10⁻² fg/test; F: 7.5 x 10⁻³ fg/test); Tc IV (G: 5.0 x 10⁻¹ fg/test, H: 5.0 x 10⁻² fg/test); Tc V (I: 1.5 x 10⁻¹ fg/test; J: 1.5 x 10⁻² fg/test); Tc VI (K: 1.0 x 10⁻¹ fg/test; L. 1.0 x 10⁻² fg/test) Right panels: Amplification plots obtained in the LAMP reaction after analyzing the samples in a Rotor Gene 3000 thermocycler. Y axis represents fluorescence and x axis represents Cts (Threshold cycles). Only the highest dilution giving amplification and the next dilution giving non detectable results are shown.</p

Clinical specimens results from LAMP test and qPCR as a comparator.

<p>Clinical specimens results from LAMP test and qPCR as a comparator.</p

Analytical sensitivity of LAMP assay in spiked EDTA blood samples.

<p>EDTA blood samples spiked with different quantities of purified <i>T</i>. <i>cruzi</i> DNA were processed using RAS columns or the Boil & Spin method. (A) EDTA blood processed using RAS columns. (B): EDTA blood processed by Boil & Spin method. Upper panels: LAMP reaction products analyzed by electrophoresis in 1.2% agarose gels and stained with ethidium bromide. Bottom panels: pictures of LAMP reaction products visualized by the naked eye.</p

INCLUSIVITY in <i>Trypanosoma cruzi</i> strains representative of different DTUs (fg/test).

<p>INCLUSIVITY in <i>Trypanosoma cruzi</i> strains representative of different DTUs (fg/test).</p